Cellular dynamics of the SecA ATPase at the single molecule level.

In bacteria, the SecA ATPase provides the driving force for protein secretion via the SecYEG translocon. While the dynamic interplay between SecA and SecYEG in translocation is widely appreciated, it is not clear how SecA associates with the translocon in the crowded cellular environment. We use super-resolution microscopy to directly visualize the dynamics of SecA in Escherichia coli at the single-molecule level. We find that SecA is predominantly associated with and evenly distributed along the cytoplasmic membrane as a homodimer, with only a minor cytosolic fraction. SecA moves along the cell membrane as three distinct but interconvertible diffusional populations: (1) A state loosely associated with the membrane, (2) an integral membrane form, and (3) a temporarily immobile form. Disruption of the proton-motive-force, which is essential for protein secretion, re-localizes a significant portion of SecA to the cytoplasm and results in the transient location of SecA at specific locations at the membrane. The data support a model in which SecA diffuses along the membrane surface to gain access to the SecYEG translocon.

Single-molecule analysis of dynamics and interactions of the SecYEG translocon.

Protein translocation and insertion into the bacterial cytoplasmic membrane are the essential processes mediated by the Sec machinery. The core machinery is composed of the membrane-embedded translocon SecYEG that interacts with the secretion-dedicated ATPase SecA and translating ribosomes. Despite the simplicity and the available structural insights on the system, diverse molecular mechanisms and functional dynamics have been proposed. Here, we employ total internal reflection fluorescence microscopy to study the oligomeric state and diffusion of SecYEG translocons in supported lipid bilayers at the single-molecule level. Silane-based coating ensured the mobility of lipids and reconstituted translocons within the bilayer. Brightness analysis suggested that approx. 70% of the translocons were monomeric. The translocons remained in a monomeric form upon ribosome binding, but partial oligomerization occurred in the presence of nucleotide-free SecA. Individual trajectories of SecYEG in the lipid bilayer revealed dynamic heterogeneity of diffusion, as translocons commonly switched between slow and fast mobility modes with corresponding diffusion coefficients of 0.03 and 0.7 µm2 ·s-1 . Interactions with SecA ATPase had a minor effect on the lateral mobility, while bound ribosome:nascent chain complexes substantially hindered the diffusion of single translocons. Notably, the mobility of the translocon:ribosome complexes was not affected by the solvent viscosity or macromolecular crowding modulated by Ficoll PM 70, so it was largely determined by interactions within the lipid bilayer and at the interface. We suggest that the complex mobility of SecYEG arises from the conformational dynamics of the translocon and protein:lipid interactions.

Bacterial coexistence driven by motility and spatial competition.

Elucidating elementary mechanisms that underlie bacterial diversity is central to ecology1,2 and microbiome research3. Bacteria are known to coexist by metabolic specialization4, cooperation5 and cyclic warfare6-8. Many species are also motile9, which is studied in terms of mechanism10,11, benefit12,13, strategy14,15, evolution16,17 and ecology18,19. Indeed, bacteria often compete for nutrient patches that become available periodically or by random disturbances2,20,21. However, the role of bacterial motility in coexistence remains unexplored experimentally. Here we show that-for mixed bacterial populations that colonize nutrient patches-either population outcompetes the other when low in relative abundance. This inversion of the competitive hierarchy is caused by active segregation and spatial exclusion within the patch: a small fast-moving population can outcompete a large fast-growing population by impeding its migration into the patch, while a small fast-growing population can outcompete a large fast-moving population by expelling it from the initial contact area. The resulting spatial segregation is lost for weak growth-migration trade-offs and a lack of virgin space, but is robust to population ratio, density and chemotactic ability, and is observed in both laboratory and wild strains. These findings show that motility differences and their trade-offs with growth are sufficient to promote diversity, and suggest previously undescribed roles for motility in niche formation and collective expulsion-containment strategies beyond individual search and survival.

Single-Molecule Studies on the Protein Translocon.

Single-molecule studies provide unprecedented details about processes that are difficult to grasp by bulk biochemical assays that yield ensemble-averaged results. One of these processes is the translocation and insertion of proteins across and into the bacterial cytoplasmic membrane. This process is facilitated by the universally conserved secretion (Sec) system, a multi-subunit membrane protein complex that consists of dissociable cytoplasmic targeting components, a molecular motor, a protein-conducting membrane pore, and accessory membrane proteins. Here, we review recent insights into the mechanisms of protein translocation and membrane protein insertion from single-molecule studies.

Steric exclusion and protein conformation determine the localization of plasma membrane transporters.

The plasma membrane (PM) of Saccharomyces cerevisiae contains membrane compartments, MCC/eisosomes and MCPs, named after the protein residents Can1 and Pma1, respectively. Using high-resolution fluorescence microscopy techniques we show that Can1 and the homologous transporter Lyp1 are able to diffuse into the MCC/eisosomes, where a limited number of proteins are conditionally trapped at the (outer) edge of the compartment. Upon addition of substrate, the immobilized proteins diffuse away from the MCC/eisosomes, presumably after taking a different conformation in the substrate-bound state. Our data indicate that the mobile fraction of all integral plasma membrane proteins tested shows extremely slow Brownian diffusion through most of the PM. We also show that proteins with large cytoplasmic domains, such as Pma1 and synthetic chimera of Can1 and Lyp1, are excluded from the MCC/eisosomes. We hypothesize that the distinct localization patterns found for these integral membrane proteins in S. cerevisiae arises from a combination of slow lateral diffusion, steric exclusion, and conditional trapping in membrane compartments.