DOI: 10.5061/dryad.bvq83bkp4
title: Data from: Extrusion-modulated DnaA activity oscillations coordinate DNA replication with biomass growth

description: 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.
description: # Data from: Extrusion-modulated DnaA activity oscillations coordinate DNA
 replication with biomass growth Dataset DOI:
 [10.5061/dryad.bvq83bkp4](https://doi.org/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 * 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 *
 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
 * 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
 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.