Geometric properties of Deinococcus radiodurans cells and nucleoids reflect effects of radiation exposure or the deletion of key genome-associated proteins

In well-studied model organisms like Escherichia coli and Bacillus subtilis, nucleoid-associated proteins (NAPs) play a pivotal role in maintaining the hierarchical organization and compaction of bacterial DNA under stress. Unusually, the extremophile Deinococcus radiodurans maintains a highly-organized and condensed nucleoid as its default state; this may contribute to D. radiodurans’ extraordinarily high tolerance of ionizing radiation (IR) and other stresses. As a class, NAPs may be a key link between nucleoid compaction and stress tolerance, but what role they play in regulating the compaction of DNA in D. radiodurans is unknown. This knowledge gap highlights the need for a screening tool to identify proteins involved in nuclear compaction and stress response. Previous studies of the D. radiodurans nucleoid relied on manual annotation and largely qualitative metrics. Here, we introduce a high-throughput approach to quantifying the geometric properties of cells and nucleoids, using confocal microscopy, digital reconstructions of bacterial cells, and machine learning. We measure pronounced changes in D. radiodurans cells and nucleoids following either IR or genetic knocking-out of candidate proteins. We find that the nucleoid becomes a more-compact and more-spherical shape as IR increases, and that cells and nucleoids adopt morphological extremes upon protein-encoding gene deletion. Thus, here we demonstrate an adaptable, quantitative methodology for analyzing D. radiodurans’ robust response to stressors and for screening candidate proteins for their role in the organization of the bacterial nucleoid.