High-throughput, subpixel precision analysis of bacterial morphogenesis and intracellular spatio-temporal dynamics.

Bacteria display various shapes and rely on complex spatial organization of their intracellular components for many cellular processes. This organization changes in response to internal and external cues. Quantitative, unbiased study of these spatio-temporal dynamics requires automated image analysis of large microscopy datasets. We have therefore developed MicrobeTracker, a versatile and high-throughput image analysis program that outlines and segments cells with subpixel precision, even in crowded images and mini-colonies, enabling cell lineage tracking. MicrobeTracker comes with an integrated accessory tool, SpotFinder, which precisely tracks foci of fluorescently labelled molecules inside cells. Using MicrobeTracker, we discover that the dynamics of the extensively studied Escherichia coli Min oscillator depends on Min protein concentration, unveiling critical limitations in robustness within the oscillator. We also find that the fraction of MinD proteins oscillating increases with cell length, indicating that the oscillator has evolved to be most effective when cells attain an appropriate length. MicrobeTracker was also used to uncover novel aspects of morphogenesis and cell cycle regulation in Caulobacter crescentus. By tracking filamentous cells, we show that the chromosomal origin at the old-pole is responsible for most replication/separation events while the others remain largely silent despite contiguous cytoplasm. This surprising position-dependent silencing is regulated by division.