Data from: Extrusion-modulated DnaA activity oscillations coordinate DNA replication with biomass growth
Data files
Oct 29, 2025 version files 478.76 MB
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Fish_data_extracted_from_microscopic_images_via_MicrobeJ.zip
478.76 MB
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README.md
4.42 KB
Abstract
Robust control of DNA replication is fundamental to bacterial proliferation. In Escherichia coli, replication initiation is thought to be regulated by oscillations in DnaA activity, driven by DnaA-chromosome interactions that differ among leading models. However, direct evidence linking these oscillations to replication initiation has been lacking, and existing models fail to explain the observed decoupling of replication initiation from dnaA expression. Here, we establish a direct link between DnaA activity and replication initiation by demonstrating robust oscillations in DnaA activity, which peak precisely at replication initiation across diverse growth conditions and genetic perturbations. Notably, these oscillations persist even when dnaA transcription remains constant, suggesting a regulatory mechanism that modulates DnaA activity independently of its expression. Additionally, we propose an extrusion model in which DNA-binding proteins sense biomass-DNA imbalance and extrude DnaA from the chromosome to trigger replication, overcoming limitations of existing models. Consistent with this model, perturbation of the nucleoid-associated protein H-NS modulates DnaA activity and replication timing, supporting its mechanistic validity.
Dataset DOI: 10.5061/dryad.bvq83bkp4
Description of the data and file structure
To analyze the cell cycle-dependent changes in dnaA transcription levels and DnaA activity, this study employed the mRNA FISH method to quantitatively analyze dnaA mRNA and lacZ mRNA—which is driven by the reporter construct Psyn66/Pcon-lacZ—under different growth conditions and genetic perturbations. For the acquired bright-field and fluorescence images, the parameters provided here, including cell morphology and fluorescence intensity, were extracted using MicrobeJ software (DOI: 10.1038/nmicrobiol 2016.77).
This dataset comprises various MATLAB .mat files in each subfolder for DnaA-activity reporter host cells cultivated under various growth conditions.
Files and variables
File: Fish_data_extracted_from_microscopic_images_via_MicrobeJ.zip
Description:
File structure information is as follows:
- wild-type MGCL1 host cells in various media.
- medium_M1
- MGCL1_M1_Pcon_lacZ.mat
- MGCL1_M1_Psyn66_lacZ.mat
- MGCL1_M1_PdnaA_dnaA.mat
- medium_M2
- MGCL1_M2_Pcon_lacZ.mat
- MGCL1_M2_Psyn66_lacZ.mat
- medium_M3
- MGCL1_M3_Pcon_lacZ.mat
- MGCL1_M3_Psyn66_lacZ.mat
- medium_M6
- MGCL1_M6_Pcon_lacZ.mat
- MGCL1_M6_Psyn66_lacZ.mat
- medium_M12
- MGCL1_M12_Pcon_lacZ.mat
- MGCL1_M12_Psyn66_lacZ.mat
- medium_M13
- MGCL1_M13_Pcon_lacZ.mat
- MGCL1_M13_Psyn66_lacZ.mat
- medium_M18
- MGCL1_M18_Pcon_lacZ.mat
- MGCL1_M18_Psyn66_lacZ.mat
- medium_M1
- dnaA-titratable (RdnaA2) host cells in M6 medium with various aTc concentration
- in aTc 2 ng/ml
- RdnaA2_M6_aTc 2_Pcon_lacZ.mat
- RdnaA2_M6_aTc 2_Psyn66_lacZ.mat
- RdnaA2_M6_aTc 2_Ptet_dnaA.mat
- in aTc 5 ng/ml
- RdnaA2_M6_aTc 5_Pcon_lacZ.mat
- RdnaA2_M6_aTc 5_Psyn66_lacZ.mat
- in aTc 8 ng/ml
- RdnaA2_M6_aTc 8_Pcon_lacZ.mat
- RdnaA2_M6_aTc 8_Psyn66_lacZ.mat
- in aTc 2 ng/ml
- hns-titratable (Rhns2) cultivated in M1 with various aTc concentrations
- in aTc 0 ng/ml
- Rhns2_M1_aTc 0_Pcon_lacZ.mat
- Rhns2_M1_aTc 0_Psyn66_lacZ.mat
- in aTc 4 ng/ml
- Rhns2_M1_aTc 4_Pcon_lacZ.mat
- Rhns2_M1_aTc 4_Psyn66_lacZ.mat
- in aTc 8 ng/ml
- Rhns2_M1_aTc 8_Pcon_lacZ.mat
- Rhns2_M1_aTc 8_Psyn66_lacZ.mat
- in aTc 16 ng/ml
- Rhns2_M1_aTc 16_Pcon_lacZ.mat
- Rhns2_M1_aTc 16_Psyn66_lacZ.mat
- in aTc 50 ng/ml
- Rhns2_M1_aTc 50_Pcon_lacZ.mat
- Rhns2_M1_aTc 50_Psyn66_lacZ.mat
- in aTc 0 ng/ml
- hns-titratable (Rhns2) cultivated in M6 with various aTc concentrations
- in aTc 0 ng/ml
- Rhns2_M6_aTc 0_Pcon_lacZ.mat
- Rhns2_M6_aTc 0_Psyn66_lacZ.mat
- in aTc 4 ng/ml
- Rhns2_M6_aTc 4_Pcon_lacZ.mat
- Rhns2_M6_aTc 4_Psyn66_lacZ.mat
- in aTc 8 ng/ml
- Rhns2_M6_aTc 8_Pcon_lacZ.mat
- Rhns2_M6_aTc 8_Psyn66_lacZ.mat
- in aTc 16 ng/ml
- Rhns2_M6_aTc 16_Pcon_lacZ.mat
- Rhns2_M6_aTc 16_Psyn66_lacZ.mat
- in aTc 50 ng/ml
- Rhns2_M6_aTc 50_Pcon_lacZ.mat
- Rhns2_M6_aTc 50_Psyn66_lacZ.mat
- in aTc 0 ng/ml
For each growth condition, we performed FISH analysis with lacZ probes on cells containing either the Psyn66-lacZ or Pcon-lacZ plasmid. Cell parameters were subsequently acquired from bright-field and fluorescence images with MicrobeJ software and are contained within the respective Psyn66.mat and Pcon.mat files.
Additionally, we analyzed the dnaA expression under specific growth conditions and performed FISH with dnaA probes. The cell parameters are saved in the RdnaA2_M6_aTc 2_Ptet_dnaA.mat and RdnaA2_M6_aTc 2_Ptet_dnaA.mat files.
Each .mat file is the original file exported from MicrobeJ. The cell's morphological parameters need to be obtained from the SHAPE property within the 'test' structure data, and the fluorescence intensity data needs to be obtained from the INTENSITY property.
-SHAPE: including morphological parameters of the cells, such as the cell area, length, width, circularity, etc.
-INTENSITY: containing the signal intensity information of the cells in the bright field, RFP field, and DAPI field channels, which are stored in rows 1, 2, and 3, respectively.
Other attributes in the test structure data, such as LOCATION, POSITION, and IMAGE, which contain information about the cell's position within the images, are not used in our study.
