Transient non-specific DNA binding dominates the target search of bacterial DNA-binding proteins.

Despite their diverse biochemical characteristics and functions, all DNA-binding proteins share the ability to accurately locate their target sites among the vast excess of non-target DNA. Toward identifying universal mechanisms of the target search, we used single-molecule tracking of 11 diverse DNA-binding proteins in living Escherichia coli. The mobility of these proteins during the target search was dictated by DNA interactions rather than by their molecular weights. By generating cells devoid of all chromosomal DNA, we discovered that the nucleoid is not a physical barrier for protein diffusion but significantly slows the motion of DNA-binding proteins through frequent short-lived DNA interactions. The representative DNA-binding proteins (irrespective of their size, concentration, or function) spend the majority (58%-99%) of their search time bound to DNA and occupy as much as ∼30% of the chromosomal DNA at any time. Chromosome crowding likely has important implications for the function of all DNA-binding proteins.

Geometry sensing by self-organized protein patterns.

In the living cell, proteins are able to organize space much larger than their dimensions. In return, changes of intracellular space can influence biochemical reactions, allowing cells to sense their size and shape. Despite the possibility to reconstitute protein self-organization with only a few purified components, we still lack knowledge of how geometrical boundaries affect spatiotemporal protein patterns. Following a minimal systems approach, we used purified proteins and photolithographically patterned membranes to study the influence of spatial confinement on the self-organization of the Min system, a spatial regulator of bacterial cytokinesis, in vitro. We found that the emerging protein pattern responds even to the lateral, two-dimensional geometry of the membrane such that, as in the three-dimensional cell, Min protein waves travel along the longest axis of the membrane patch. This shows that for spatial sensing the Min system does not need to be enclosed in a three-dimensional compartment. Using a computational model we quantitatively analyzed our experimental findings and identified persistent binding of MinE to the membrane as requirement for the Min system to sense geometry. Our results give insight into the interplay between geometrical confinement and biochemical patterns emerging from a nonlinear reaction-diffusion system.

Stationary Patterns in a Two-Protein Reaction-Diffusion System.

Patterns formed by reaction-diffusion mechanisms are crucial for the development or sustenance of most organisms in nature. Patterns include dynamic waves, but are more often found as static distributions, such as animal skin patterns. Yet, a simplistic biological model system to reproduce and quantitatively investigate static reaction-diffusion patterns has been missing so far. Here, we demonstrate that the Escherichia coli Min system, known for its oscillatory behavior between the cell poles, is under certain conditions capable of transitioning to quasi-stationary protein distributions on membranes closely resembling Turing patterns. We systematically titrated both proteins, MinD and MinE, and found that removing all purification tags and linkers from the N-terminus of MinE was critical for static patterns to occur. At small bulk heights, dynamic patterns dominate, such as in rod-shaped microcompartments. We see implications of this work for studying pattern formation in general, but also for creating artificial gradients as downstream cues in synthetic biology applications.

Surface topology assisted alignment of Min protein waves.

Self-organization of proteins into large-scale structures is of pivotal importance for the organization of cells. The Min protein system of the bacterium Escherichia coli is a prime example of how pattern formation occurs via reaction-diffusion. We have previously demonstrated how Min protein patterns are influenced by compartment geometry. Here we probe the influence of membrane surface topology, as an additional regulatory element. Using microstructured membrane-clad soft polymer substrates, Min protein patterns can be aligned. We demonstrate that Min pattern alignment starts early during pattern formation and show that macroscopic millimeter-sized areas of protein patterns of well-defined orientation can be generated.