Subcellular localization and dynamics of transmembrane and membrane associated proteins in Corynebacterium glutamicum

The cytoplasmic membrane of Corynebacterium glutamicum comprises 660 predicted membrane integral proteins of various functional categories, including transport, signal transduction, protein translocation, proteolysis and energy metabolism. Being an important industrial producer of amino acids, studying the membrane proteome of this organism is relevant for a better understanding of its fundamental processes and possible targeted optimizations. Here, we analyzed the subcellular localization of different transmembrane and membrane-associated proteins of Corynebacterium glutamicum, including the fructose specific PtsF and the glucose specific PtsG transporters, the respiratory chain complex II component SdhA, the small-conductance mechanosensitive channel YggB, and the polar/septa scaffold DivIVA. Using widefield and single molecule localization microscopy, we discovered that PtsG and PtsF form membrane embedded clusters with no defined positions. A similar pattern was observed for SdhA, which curiously, seem to colocalize with PtsF in our widefield microscopy experiments. Size, number and fluorescence of the observed PTS clusters change upon presence or absence of the transported substrate. In presence of the transport substrate, clusters significantly increased in size. Photo-activated localization microscopy (PALM) data revealed that, in presence of different carbon sources, the number of EII proteins per cluster remains the same, however the density of these clusters reduces. Our work reveals a simple mechanism for efficient membrane occupancy regulation. Clusters of PTS EII transporters are densely packed in absence of a suitable substrate. In presence of a transported substrate, the EII proteins in individual clusters occupy larger membrane areas. This mechanism allows for efficient use of the limited membrane space under varying growth conditions without need of protein removal and re-synthesis. Clustering was also observed in YggB, that localized in a punctate pattern of different intensities at the membrane. By changing the NaCl concentration in medium, ranging from 0 mM to 170 mM, we could observe C. glutamicum cells under different degrees of osmotic stress, ranging from hypoosmotic to low hyperosmotic. The extreme studied treatments resulted in impaired growth, which suggests that the cells were indeed under constant osmotic stress. Under such conditions, no changes in YggB-mNeonGreen foci number or fluorescence were observed, hinting that yggB expression is constitutive and that prolonged exposure to osmotic stress does not result in the recruitment of more channels to the membrane. Finally, we studied the dynamics of DivIVA in cells of various shapes, including L-form cells with different morphologies by performing widefield microscopy and fluorescence recovery after photobleaching (FRAP) experiments with a strain expressing DivIVA-mCherry. DivIVA is a membrane-associated protein that serves as a scaffold for different proteins related to cell division and cell growth. Cell division in bacterial L-forms is unlike the coordinated and precise division observed in cell-walled bacteria. It takes place randomly, not necessarily linked to the divisome and is highly influenced by perturbations in the surrounding environment. Due to this, it is not uncommon to find L-form cells with multiple, parts of, or no chromosomes. Still, in these cells, DivIVA continues to localize in regions of negative curvatures, characterized as bumps in the cytoplasmic membrane. In completely spherical cells, with no regions of higher negative curvature, DivIVA localize as larger foci. Our FRAP results demonstrated that in regions of bumps, the diffusion rate of DivIVA-mCherry is similar to the one observed for poles of rod-shaped cells, but with a significant lower half-time recovery. Furthermore, the diffusion rate on smooth surfaces was more than double, and the half-time recovery less half as the one observed in bumps. This suggests that DivIVA is significantly more mobile in such regions. Moreover, using novel microscopy techniques, the work presented on this thesis shed a light on unexplored facets of membrane protein compartmentalization in Corynebacterium glutamicum.