Escherichia coli segregates protein aggregates to the poles by nucleoid exclusion. Combined with cell divisions, this generates heterogeneous aggregate distributions in subsequent cell generations. We studied the robustness of this process with differing medium richness and antibiotics stress, which affect nucleoid size, using multimodal, time-lapse microscopy of live cells expressing both a fluorescently tagged chaperone (IbpA), which identifies in vivo the location of aggregates, and HupA-mCherry, a fluorescent variant of a nucleoid-associated protein. We find that the relative sizes of the nucleoid's major and minor axes change widely, in a positively correlated fashion, with medium richness and antibiotic stress. The aggregate's distribution along the major cell axis also changes between conditions and in agreement with the nucleoid exclusion phenomenon. Consequently, the fraction of aggregates at the midcell region prior to cell division differs between conditions, which will affect the degree of asymmetries in the partitioning of aggregates between cells of future generations. Finally, from the location of the peak of anisotropy in the aggregate displacement distribution, the nucleoid relative size, and the spatiotemporal aggregate distribution, we find that the exclusion of detectable aggregates from midcell is most pronounced in cells with mid-sized nucleoids, which are most common under optimal conditions. We conclude that the aggregate management mechanisms of E. coli are significantly robust but are not immune to stresses due to the tangible effect that these have on nucleoid size. IMPORTANCEEscherichia coli segregates protein aggregates to the poles by nucleoid exclusion. From live single-cell microscopy studies of the robustness of this process to various stresses known to affect nucleoid size, we find that nucleoid size and aggregate preferential locations change concordantly between conditions. Also, the degree of influence of the nucleoid on aggregate positioning differs between conditions, causing aggregate numbers at midcell to differ in cell division events, which will affect the degree of asymmetries in the partitioning of aggregates between cells of future generations. Finally, we find that aggregate segregation to the cell poles is most pronounced in cells with mid-sized nucleoids. We conclude that the energy-free process of the midcell exclusion of aggregates partially loses effectiveness under stressful conditions.
The fast adaptation of Escherichia coli to stressful environments includes the regulation of gene expression rates, mainly of transcription, by specific and global stress-response mechanisms. To study the effects of mechanisms acting on a global level, we observed with single molecule sensitivity the effects of mild acidic shift and oxidative stress on the in vivo transcription dynamics of a probe gene encoding an RNA target for MS2d-GFP, under the control of a synthetic promoter. After showing that this promoter is uninvolved in fast stress-response pathways, we compared its kinetics of transcript production under stress and in optimal conditions. We find that, following the application of either stress, the mean rates of transcription activation and of subsequent RNA production of the probe gene are reduced, particularly under oxidative stress. Meanwhile, the noise in RNA production decreases under oxidative stress, but not under acidic shift. From distributions of intervals between consecutive RNA productions, we infer that the number and duration of the rate-limiting steps in transcription initiation change, following the application of stress. These changes differ in the two stress conditions and are consistent with the changes in noise in RNA production. Overall, our measurements of the transcription initiation kinetics of the probe gene indicate that, following sub-lethal stresses, there are stress-specific changes in the dynamics of transcription initiation of the probe gene that affect its mean rate and noise of transcript production. Given the non-involvement of the probe gene in stress-response pathways, we suggest that these changes are caused by global response mechanisms of E. coli to stress.
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