Subcellular Dynamics of a Conserved Bacterial Polar Scaffold Protein.

In order to survive, bacterial cells rely on precise spatiotemporal organization and coordination of essential processes such as cell growth, chromosome segregation, and cell division. Given the general lack of organelles, most bacteria are forced to depend on alternative localization mechanisms, such as, for example, geometrical cues. DivIVA proteins are widely distributed in mainly Gram-positive bacteria and were shown to bind the membrane, typically in regions of strong negative curvature, such as the cell poles and division septa. Here, they have been shown to be involved in a multitude of processes: from apical cell growth and chromosome segregation in actinobacteria to sporulation and inhibition of division re-initiation in firmicutes. Structural analyses revealed that DivIVA proteins can form oligomeric assemblies that constitute a scaffold for recruitment of other proteins. However, it remained unclear whether interaction with partner proteins influences DivIVA dynamics. Using structured illumination microscopy (SIM), single-particle tracking (SPT) microscopy, and fluorescent recovery after photobleaching (FRAP) experiments, we show that DivIVA from Corynebacterium glutamicum is mobilized by its binding partner ParB. In contrast, we show that the interaction between Bacillus subtilis DivIVA and its partner protein MinJ reduces DivIVA mobility. Furthermore, we show that the loss of the rod-shape leads to an increase in DivIVA dynamics in both organisms. Taken together, our study reveals the modulation of the polar scaffold protein by protein interactors and cell morphology. We reason that this leads to a very simple, yet robust way for actinobacteria to maintain polar growth and their rod-shape. In B. subtilis, however, the DivIVA protein is tailored towards a more dynamic function that allows quick relocalization from poles to septa upon division.

Elongation factor P is required for EIIGlc translation in Corynebacterium glutamicum due to an essential polyproline motif.

Translating ribosomes require elongation factor P (EF-P) to incorporate consecutive prolines (XPPX) into nascent peptide chains. The proteome of Corynebacterium glutamicum ATCC 13032 contains a total of 1,468 XPPX motifs, many of which are found in proteins involved in primary and secondary metabolism. We show here that synthesis of EIIGlc , the glucose-specific permease of the phosphoenolpyruvate (PEP): sugar phosphotransferase system (PTS) encoded by ptsG, is strongly dependent on EF-P, as an efp deletion mutant grows poorly on glucose as sole carbon source. The amount of EIIGlc is strongly reduced in this mutant, which consequently results in a lower rate of glucose uptake. Strikingly, the XPPX motif is essential for the activity of EIIGlc , and substitution of the prolines leads to inactivation of the protein. Finally, translation of GntR2, a transcriptional activator of ptsG, is also dependent on EF-P. However, its reduced amount in the efp mutant can be compensated for by other regulators. These results reveal for the first time a translational bottleneck involving production of the major glucose transporter EIIGlc , which has implications for future strain engineering strategies.

Substrate-dependent cluster density dynamics of Corynebacterium glutamicum phosphotransferase system permeases.

Many bacteria take up carbohydrates by membrane-integral sugar specific phosphoenolpyruvate-dependent carbohydrate:phosphotransferase systems (PTS). Although the PTS is centrally involved in regulation of carbon metabolism in different bacteria, little is known about localization and putative oligomerization of the permease subunits (EII). Here, we analyzed localization of the fructose specific PtsF and the glucose specific PtsG transporters, as well as the general components EI and HPr from Corynebacterium glutamicum using widefield and single molecule localization microscopy. PtsF and PtsG form membrane embedded clusters that localize in a punctate pattern. Size, number and fluorescence of the membrane clusters change upon presence or absence of the transported substrate, and a direct influence of EI and HPr was not observed. In presence of the transport substrate, EII clusters significantly increased in size. Photo-activated localization microscopy 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.