Coordination of biomass growth and DNA replication through replication initiation and DnaA activity oscillations.

(A) Schematic representation of the discrepancy between biomass accumulation and DNA replication. While biomass grows exponentially, DNA synthesis progresses linearly, necessitating replication initiation events to maintain coordination. (B) Mechanistic model for biomass-DNA coordination in bacteria. A molecular sensor detects deviations between cell mass and DNA content, transmitting this information to regulatory controllers that compensate by either increasing DNA replication or restricting biomass accumulation. (C) Illustration of cyclic DnaA activity oscillations aligning with replication initiation to ensure precise cell cycle control.

Construction and characterization of the dnaA-titratable strain.

(A) Schematic of the dnaA-titratable strain. A dnaA gene under the control of the Ptet promoter was inserted near oriC and regulated by a Ptet-tetR feedback loop integrated at the intS locus, enabling fine-tuned expression control. The native dnaA gene was replaced with a kanamycin resistance cassette (kanr). (B–F) Characterization of dnaA-titratable cells (red open circles) grown in rich defined medium with glycerol (M6) under varying aTc concentrations. Measured parameters include dnaA mRNA levels (B), growth rate (C), population-averaged cellular mass (D), population-averaged oriC numbers (E), and initiation mass (F). Wild-type MG1655 values (black triangles) are shown for comparison. dnaA mRNA levels, normalized to wild-type MG1655, increased with aTc concentration from 0.75 to 20 ng·ml−1. Cellular mass was determined by OD600 divided by cell number concentration. oriC copy numbers were measured using a run-out assay, and initiation mass was calculated as the ratio of cellular mass to oriC numbers. Data represent means ± SD (n = 5 biological replicates). (G) Relationship between relative initiation mass and relative dnaA mRNA levels, compared with predictions from the initiation titration model (blue line) and the switch model (purple line). Experimental data are overlaid for validation.

Development of a DnaA activity reporter system.

(A) Schematic of promoter design and screening. Sixty-seven synthetic promoters were constructed by inserting various DnaA-boxes around the promoter core to drive gfp expression in a dnaA-titratable strain, where dnaA expression was regulated by aTc concentration. After pre-cultivation, GFP fluorescence per OD600 was measured using a microplate reader in cells grown under low (0.5 ng·ml−1) or high (50 ng·ml−1) aTc concentrations to determine repression fold-change. (B) Repression fold-change of synthetic promoters. Pcon (a promoter lacking DnaA-boxes) and Pnative (the endogenous dnaA promoter) served as negative and positive controls, shown in gray and yellow, respectively. (C) Response curves of three promoters to varying dnaA expression levels, with their promoter architectures shown on the right. Promoter activity was assessed by relative gfp mRNA levels, normalized to the lowest dnaA expression condition. Schematic (D) and response curves (E) of Psyn66 and Pnative responses to SeqA in the seqA-titratable strain. Promoter activity was quantified from gfp transcript levels in seqA-titratable cells containing the Psyn66-GFP plasmid, normalized to the lowest seqA expression level. All cells were grown in rich defined medium supplemented with glycerol across different aTc concentrations. Data represent mean ± SD from 3 biological replicates (B,C,E).

DnaA activity oscillations decoupled from dnaA transcription fluctuations.

(A) Representative lacZ mRNA FISH images of MG1655 Δlac cells transformed with lacZ expression plasmids driven by Psyn66 (DnaA-boxes around promoter core), Pcon (no DnaA-box around promoter core), or Pneg (mutated promoter core). Yellow outlines indicate cell boundaries identified from phase-contrast images. (B) Relative lacZ mRNA concentrations driven by Psyn66 (left) and Pcon (right) across different cell volumes. Relative concentrations were determined from volume-specific lacZ mRNA fluorescence intensities, normalized to the population average. Volume-binned data for Psyn66 ([mZ] (Psyn66)) and Pcon ([mZ] (Pcon)) are shown as open circles and were used to calculate ksyn66. (C) Schematic of a strain with autoregulated dnaA transcription carrying a DnaA activity reporter plasmid. Cell cycle-dependent fluctuations in relative DnaA activity (D) and relative dnaA mRNA concentrations (E) in cells from panel C, grown in rich defined medium supplemented with glucose. Relative DnaA activity , calculated from volume-binned data in panel B, was smoothed and plotted as a red curve (D). Relative dnaA mRNA concentrations were determined from volume-specific dnaA mRNA fluorescence intensities, normalized to the population average, with volume-binned data shown as open circles (E). (F–H) Same as panels C–E, but for cells with aTc induced dnaA transcription, grown in rich defined medium supplemented with glycerol under 2 ng·ml−1 aTc induction. More than 8,000 cells were analyzed per growth condition, and all error bars correspond to standard error of the mean (SEM).

Tight correlation between DnaA activity oscillations and DNA replication initiation.

(A) Cell cycle-dependent DnaA activity oscillations in wild-type cells across various growth conditions. DnaA activity is represented by . Volume-binned DnaA activity (red circles) was smoothed and plotted as a red curve. The representative birth-to-division cell cycle, defined as the cell volume doubling interval containing the majority of cells, is shaded in gray. The vertical line indicates the cell volume at replication initiation (Vi). (B) Cell cycle-dependent DnaA activity oscillations in dnaA-titratable cells grown in M6 medium with varying aTc concentrations. More than 8,000 cells were analyzed per growth condition, and error bars show the mean ± SEM. (C) Correlation between V∗ and Vi in wild-type (squares) and dnaA-titratable (circles) cells. V∗ represents the cell volume at the peak of DnaA activity within the representative birth-to-division cell cycle. The black line indicates equivalence between V∗ and Vi.

An extrusion model explains DnaA shut-down dynamics.

(A) Genetic circuit of the deactivated CRISPR-Cas system for dnaA transcription shut-down. dnaA gene is targeted by a constitutively expressed sgRNA, while dUn1Cas12f1 expression is inhibited by TetR repressor. These transcription units are separated by terminators. The cassette was integrated into the chromosome near the oriC locus. DnaA shutdown is induced by the addition of aTc. (B) Time-course of relative dnaA mRNA levels (red line, left axis) and total oriC number (green line, right axis) following the addition of 50 ng·ml−1 aTc at time 0 (dashed line). dnaA mRNA levels were normalized to wild-type levels, and oriC numbers were normalized to their initial values. Error bars indicate mean ± SD (n = 3 biologically independent experiments). (C) Predicted increases in total oriC number during dnaA transcription shut-down based on three models: the titration model, switch model, and extrusion model. Shutdown was simulated by setting dnaA transcription to zero at time 0 (dashed line). (D) Schematic of the extrusion model. The model introduces extruder(s) as additional regulators of biomass-DNA coordination, complementing the role of DnaA (left). Increased binding of the extruder to DNA promotes the release of DnaA from DnaA-boxes (right). (E) Comparison of the relationship between relative initiation mass and relative dnaA mRNA levels from experimental data (Fig. 2F) and predictions of the extrusion model.

Titration of hns expression modulates DnaA activity and replication initiation.

(A) Genetic circuit of the hns-titratable strain. Expression of hns is controlled by a Ptet-tetR negative feedback loop integrated at the attB site, with the native hns coding sequence replaced by a kanamycin resistance gene. The plasmid contains Psyn66-mcherry and Pcon-gfp expression cassettes was used to assess DnaA activity. DnaA activity (B) and initiation mass (C) were characterized in M6 medium with varying hns expression levels during steady-state cultivation. (D) mRNA levels of hns (green circles) and dnaA (blue squares) relative to wild-type levels, along with DnaA activity (orange rhombus), were measured during hns shift-up. hns shift-up was induced by the addition of 50 ng·ml−1 aTc at time 0 (dashed line), following steady-state cultivation in M6 medium without aTc. (E) Dynamics of population-averaged oriC number (blue rhombus, left axis) and cellular mass (gray rhombus, right axis) during hns shift-up. Data represent mean ± SD from 3 biological replicates (B-E). (F) Cell cycle-dependent DnaA activity oscillations in hns-titratable cells cultivated in M1 and M6 media under varying aTc concentrations. Relative hns mRNA levels are indicated for each condition. More than 8,000 cells were analyzed for each condition and error bars show the mean ± SEM. (G) Correlation between the volume at maximal DnaA activity (V∗) and the volume at replication initiation (Vi) for hns-titratable cells grown in M1 (green down-pointing triangle) and M6 (red up-pointing triangle) media with varying hns expression levels. Data for dnaA-titratable cells (gray circles) and wild-type dnaA-autoregulated cells (Fig. 5C) are included for comparison.