Dynamic plasmid clustering in Bacteria: Influence of transcriptional activity and host cellular states

Abstract

High-copy-number (hcn) plasmids exhibit distinct spatial organization within bacterial cells, yet the underlying mechanisms and functional implications remain poorly understood. In this study, we systematically investigated the role of transcriptional activity and host cellular states in plasmid clustering. Using fluorescence imaging and quantitative analyses, we observed that transcriptional repression induced transient plasmid clustering, while transcriptional enhancement influenced clustering in a promoter-dependent manner. Additionally, environmental cues, such as glucose addition and growth phase transitions, modulate plasmid clustering independently of transcriptional activity. Glucose supplementation led to rapid plasmid dispersion, suggesting a link between metabolic state and plasmid dynamics. The switching between dispersed and clustered plasmid distributions as growth phase changes correlate with alterations in nucleoid organization. These findings highlight the complex interplay between transcription, nucleoid structure, and cellular state in regulating plasmid spatial distribution, providing new insights into bacterial genome organization and adaptation.

Keywords: Fluorescence imaging; Plasmid clustering; Spatial organization; Transcriptional regulation.

Fig. 1

Visualization and analysis of plasmid and nucleoid distributions. (A) Schematic of the fluorescence labeling system. Plasmids are labeled using a self-regulated FROS with the lacO/LacI^adi-mCherry pair, while the nucleoid is labeled with Hu-α-mTurquoise. The inset shows a representative image of plasmids (red), the nucleoid (cyan), and the cell shapes (gray; phase contrast). (B) Representative images of plasmid and nucleoid distributions with varying overlap coefficients. (C, D) Examples of normalized and centered Ripley's K functions ($\tilde{K}$) and corresponding images, illustrating different patterns of plasmid distribution. In (B) and (D), the cell outlines (white) are from corresponding phase contrast images. Scale bars equal 2 μm.

Fig. 2

Response of plasmid clustering to varying transcription conditions. (A) Schematics of transcription repression on the antibiotic resistance operon. (B) Proportions of dim cells (top panel) and time traces of average fluorescence intensities (bottom panel) in single cells under different conditions: without arabinose induction (gray), with arabinose at final concentrations of 0.2 %, 0.02 %, and 0.005 % (from dark to bright green), and with 0.2 % glucose (red). Data points and error bars represent the mean ± 1.5 × standard error. Lines serve as visual guides. Arrows in the bottom panel indicate the time points of sugar addition (gray) and the estimated time of full repression (green) based on a piecewise function fit for each arabinose concentration. (C) ${\tilde{K}}_{480}$ values of plasmid distributions following arabinose induction at different concentrations. (D) Probability distributions of OVL between plasmids and nucleoids in single cells after arabinose induction. Solid lines and error bars indicate the median and 95 % confidence interval, while dashed lines represent the quartiles. (E, F) Schematic representations of the applied promoters, including two core promoters (P_CM and P_qacR) with different numbers of enhancer sites, and the corresponding ${\tilde{K}}_{480}$ values of plasmids carrying these promoters. In the ${\tilde{K}}_{480}$ plots, data points and error bars represent the median and 95 % confidence interval. Approximately 2000 cells were analyzed for fluorescence intensity measurements, and 200 cells from three independent experiments for plasmid distribution per condition and time point. ∗P < 0.05 and ∗∗P < 0.01 by two-sided Mann-Whitney U test.

Fig. 3

Response of plasmid clustering to varying cellular states of the host cells. (A) Representative time traces of ${\tilde{K}}_{480}$ values in individual cells following glucose addition at t = 0. ${\tilde{K}}_{480}$ values of plasmid distribution (B) and the probability distributions of the overlap coefficient (OVL) between plasmids and nucleoids (C) in single cells after glucose addition. (D, E) ${\tilde{K}}_{480}$ values, the probability distributions of OVL in single cells, and the estimated cell division rate (solid line) at different cell concentrations during culture. In (B)–(E), figure elements are presented as described in Fig. 2. Approximately 200 cells from three independent experiments were analyzed per condition and time point.