Robustness of the Process of Nucleoid Exclusion of Protein Aggregates in Escherichia coli

Abstract

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.

Systematic and general method for quantifying localization in microscopy images

“…Local concentrations may be higher or lower in some cell regions depending on: (i) sites of production and degradation; (ii) diffusion; (iii) kinetics of association and dissociation with cellular structures, e.g. nucleus, cell membrane or cytoskeleton; and (iv) attraction to or exclusion from cell regions (Neeli-Venkata et al, 2016; Mondal et al, 2011). With this in mind, colocalization, anti-colocalization and non-colocalization should be considered as the relationship in the local concentrations of two types of molecules, which may be due to many factors.…”

“…This may be due to common sites of production, action, binding, or degradation. Anti-colocalization indicates two molecules have high concentrations in different cell regions and thus at least one mechanism is causing the molecules to be recruited to and/or exclude from different regions, one molecule excludes the other from a region (Bakshi et al, 2015; Neeli-Venkata et al, 2016) or the molecules eliminate each other in the same location, e.g. when non-coding RNAs binding to mRNAs both are destroyed (De Lay et al, 2013).…”

Bacterial Inclusion Bodies: Discovering Their Better Half

“…There are claims that transport of aggregates to the poles is energy-driven as studies on reassembly of pressure dissociated IBs revealed that confinement to the pole is caused by the presence of the nucleoid but reassembly of smaller aggregates into large IBs does not occur in energy and nutrient depleted cells [16]. However, there are also reports stating that exclusion of aggregates from mid-cell to the pole is energy-free, and simply results from nucleoid exclusion [17,20,[28][29][30]. Restriction of aggregates to the pole was also observed during treatment with substances disrupting the protein motive force, suggesting an energy independent process of aggregation [20].…”

Modified Pearson correlation coefficient for two-color imaging in spherocylindrical cells

“…It may also suggest common sites of production, action, or degradation. Negative correlation may suggest a physical or biochemical mechanism that sequesters red and green species from each other [ 1 , 2 ]. A number of different procedures for assessing co-localization between two images are described in a recent review [ 3 ].…”