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, that precisely tracks foci of fluorescently labeled 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.

Eukaryotic cells spatially organize mRNA processes such as translation and mRNA decay. Much less is clear in bacterial cells where the spatial distribution of mature mRNA remains ambiguous. Using a sensitive, quantitative fluorescence in situ hybridization based-method, we show here that in Caulobacter crescentus and Escherichia coli, chromosomally-expressed mRNAs largely display limited dispersion from their site of transcription during their lifetime. We estimate apparent diffusion coefficients at least 2 orders of magnitude lower than expected for freely diffusing mRNA, and provide evidence in C. crescentus that this mRNA localization restricts ribosomal mobility. Furthermore, C. crescentus RNase E appears associated with the DNA independently of its mRNA substrates. Collectively, our findings reveal that bacteria can spatially organize translation and potentially mRNA decay by using the chromosome layout as a template. This chromosome-centric organization has important implications for cellular physiology and for our understanding of gene expression in bacteria.

In vivo measurements of the mobility and binding kinetics of cellular components are essential to fully understand the biochemical processes occurring inside cells. Here, we describe a fluorescence recovery after photobleaching-based method that can be easily implemented to the study of reaction-diffusion processes in live bacteria despite their small size. We apply this method to provide new, to our knowledge, quantitative insight into multiple aspects of the bacterial translation cycle by measuring the binding kinetics and the micrometer-scale diffusive properties of the 50S ribosomal subunit in live Caulobacter cells. From our measurements, we infer that 70% of 50S subunits are engaged in translation and display, on average, limited motion on the micrometer scale, consistent with little mixing of transcripts undergoing translation. We also extract the average rate constants for the binding of 50S subunits to 30S initiation complexes during initiation and for their release from mRNAs when translation is completed. From this, we estimate the average time of protein synthesis and the average search time of 50S subunits before they engage in the next initiation event. Additionally, our experiments suggest that so-called free 50S subunits do not diffuse freely; instead their mobility is significantly slowed down, possibly through transient associations with mRNA.