Substrate-dependent cluster density dynamics in bacterial phosphotransferase system permeases

Bacteria take up carbohydrates by membrane-integral sugar specific phosphoenolpyruvate-dependent carbohydrate:phosphotransferase systems (PTS). Although PTS is at the heart of bacterial carbon uptake and centrally involved in regulation of carbon metabolism, little is known about localization and putative oligomerization of the permease subunits (EII) of PTS. Here, we analyzed localization of the fructose specific PtsF and the glucose specific PtsG transporters from C. glutamicum using widefield and single molecule localization microscopy. PtsG and PtsF form membrane embedded clusters that localize in a punctate pattern within the cell membrane. The size, number and fluorescence of the observed clusters changes 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 protein events per cluster remain the same, however the density of PTS molecules within a cluster 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 transport substrate the EII proteins in individual clusters occupy larger membrane areas, thereby decreasing protein density in individual clusters. This mechanism allows for efficient use of the limited membrane space under varying growth conditions without need of protein removal and re-synthesis. Importance The carbohydrate transport system PTS is centrally involved in the regulation of sugar metabolism. Although much is known about the regulatory interaction, the genetic control and the structure/function relationship of the individual PTS components, we know almost nothing about the spatio-temporal organization of the PTS proteins within the cell. We find dynamic clustering of PTS permeases in Corynebacterium glutamicum. Using single molecule resolution photo-activated localization microscopy we could show that PTS EII protein cluster are dynamically changing protein density upon substrate availability. Our findings imply a novel strategy of regulating limited membrane space efficiently. Furthermore, these data will provide important insights in modelling carbohydrate fluxes in cells, since current models assume a homogeneous distribution of PTS permeases within the membrane.