DOI: 10.5061/dryad.9s4mw6mqg
title: Progressive decline in old pole gene expression signal enhances phenotypic heterogeneity in bacteria

description: Cell growth and gene expression are heterogeneous processes at the
 single-cell level, leading to the emergence of multiple physiological
 states within bacterial populations. Cellular aging is a known
 deterministic driver of growth asymmetry, however its role on gene
 expression heterogeneity remains elusive. Here we show that aging mother
 cells undergo a progressive decline in old pole activity, generating
 asymmetry in product partitioning, gene expression, and cell morphology.
 We demonstrate that mother cells, when compared to their daughters,
 exhibit lower product inheritance and gene expression rates independently
 of promoter dynamics. The declining activity of maternal old poles
 generates gene expression gradients that manifest as mother-daughter
 asymmetry upon division, showing that asymmetry is built over time within
 the maternal intracellular environment. Moreover, old pole aging
 correlates with a gradual increase in cell length, leading to
 morphological asymmetry. These findings provide further evidence for aging
 as a mechanism to enhance phenotypic heterogeneity in bacterial
 populations, with possible consequences for stress response and survival.
description: The dataset contains processed data on single-cell microscopy
 observations of <em>Escherichia coli</em> MG1655 bacteria
 growing in the mother machine microfluidic device (Wang et al., 2010).
 These cells contain a fluorescent transcriptional reporter of RpoS
 expression (Zaslaver et al., 2006) or constitutive GFP expression, and
 were imaged in phase contrast (2 min intervals), GFP fluorescence (10 min
 intervals) and RFP fluorescence (10 min intervals, for cell lysis
 visualization with propidium iodide). Mother and daughter cells were
 tracked for 72h (100 generations) in M9 medium at 37°C. Raw images were
 processed using DeLTA, a pipeline for image segmentation using machine
 learning algorithms (Lugagne et al., 2020) and the data presented here was
 extracted from single-cell measurements of length at birth (right after
 division), length at division, time of birth, generations elapsed,
 elongation rates, etc. The dataset includes the code necessary to
 reproduce figures in the manuscript. Data files that include both mother
 and daughter cell information are organized as a list of two separate data
 frames, with each row referring to one generation.
description: # Data from: Progressive decline in old pole gene expression signal
 enhances phenotypic heterogeneity in bacteria --- The dataset contains
 single-cell microscopy measurements of Escherichia coli bacteria grown in
 microfluic devices (mother machine design) for 72h, with M9 media at 37°C.
 Phase contrast images were obtained in 2 min intervals, while fluorescence
 images were taken every 10 min. Images were processed with DeLTA, a
 machine learning algorithm for automated cell segmentation. The data we
 provide is the result of this processing, structured to reproduce the
 figures of the linked manuscript. It contains information on single-cell
 growth (length, elongation rates), and fluorescence measurements indicate
 the mean intensity of GFP expressed under a plasmidial reporter of RpoS
 transcription or a constitutively expressed promoter (PA1). ## Description
 of the data and file structure Each data file (.rds) corresponds to a
 script file (.R), organized according to the figure numbering in the
 linked preprint manuscript. The correspondence between figure panels,
 datasets and associated scripts is provided in the Metadata file.
 Data_Figure 1_Constitutive cells.rds: List of 2 elements, where the first
 contains mother cells and the second the daughters. Each element is a data
 frame of 32,399 rows and 8 columns, containing paired data for every cell
 division. Each row shows data of one generation. Columns include the cell
 lineage number (mat.well), time of birth (mat.time), mean fluorescence at
 birth (GFP), normalized fluorescence (normalized.GFP), elongation rates
 (elong.rates), promoter actitivy (promoter), normalized promoter actitivy
 (normalized.promoter), and standard deviation of intracellular
 fluorescence at birth (GFPsd). Data_Figure 1_RpoS cells.rds: List of 2
 elements, where the first contains mother cells and the second the
 daughters. Each element is a data frame of 21,748 rows and 8 columns,
 containing paired data for every cell division. Each row shows data of one
 generation. Columns include the cell lineage number (mat.well), time of
 birth (mat.time), mean fluorescence at birth (GFP), normalized
 fluorescence (normalized.GFP), elongation rates (elong.rates), promoter
 actitivy (promoter), normalized promoter actitivy (normalized.promoter),
 and standard deviation of intracellular fluorescence at birth (GFPsd).
 Data_Figure 1_RpoS heatmap.rds: Matrix of mother cell RpoS mean
 fluorescence measurements over time. Each row is a different mother cell,
 and each column is the mean fluorescence at a given time point (10 min
 intervals). Data_Figure 1_Constitutive heatmap.rds: Matrix of mother cell
 constitutive mean fluorescence measurements over time. Each row is a
 different mother cell, and each column is the mean fluorescence at a given
 time point (10 min intervals). Data_Figure 2_Constitutive population.rds:
 List of 2 elements, where the first contains mother cells and the second
 the daughters. Each element is a data frame of 32,399 rows and 8 columns,
 containing paired data for every cell division. Each row shows data of one
 generation. Columns include time of birth (time), mean fluorescence at
 birth (fluorescence), promoter actitivy (promoter), normalized
 fluorescence (norm.fluorescence), normalized promoter actitivy
 (norm.promoter), elongation rates (elong.rates), and standard deviation of
 intracellular fluorescence at birth (cv). Data_Figure 2_RpoS
 population.rds: List of 2 elements, where the first contains mother cells
 and the second the daughters. Each element is a data frame of 21,748 rows
 and 8 columns, containing paired data for every cell division. Each row
 shows data of one generation. Columns include time of birth (time), mean
 fluorescence at birth (fluorescence), promoter actitivy (promoter),
 normalized fluorescence (norm.fluorescence), normalized promoter actitivy
 (norm.promoter), elongation rates (elong.rates), and standard deviation of
 intracellular fluorescence at birth (cv). Data_Figure 4A_ConstGFP_daughter
 transects_cropped.rds: Daughter cell data. Measurements taken at birth.
 List of 104 elements, where each element is a matrix of fluorescence
 measurements across cell lengths. Each list element stands for a
 generation, and each matrix row is a cell measured at that generation.
 Columns are fluorescence levels at a given intracellular localization,
 from old pole to new pole. Lengths were cropped to exclude 10px off each
 side, then normalized into 20 longitudinal bins. Data_Figure
 4A_ConstGFP_mother transects_cropped.rds: Mother cell data. Measurements
 taken at birth. List of 104 elements, where each element is a matrix of
 fluorescence measurements across cell lengths. Each list element stands
 for a generation, and each matrix row is a cell measured at that
 generation. Columns are fluorescence levels at a given intracellular
 localization, from old pole to new pole. Lengths were cropped to exclude
 10px off each side, then normalized into 20 longitudinal bins. Data_Figure
 4B_RpoS_daughter transects_cropped.rds: Daughter cell data. Measurements
 taken at birth. List of 109 elements, where each element is a matrix of
 fluorescence measurements across cell lengths. Each list element stands
 for a generation, and each matrix row is a cell measured at that
 generation. Columns are fluorescence levels at a given intracellular
 localization, from old pole to new pole. Lengths were cropped to exclude
 10px off each side, then normalized into 20 longitudinal bins. Data_Figure
 4B_RpoS_mother transects_cropped.rds: Mother cell data. Measurements taken
 at birth. List of 109 elements, where each element is a matrix of
 fluorescence measurements across cell lengths. Each list element stands
 for a generation, and each matrix row is a cell measured at that
 generation. Columns are fluorescence levels at a given intracellular
 localization, from old pole to new pole. Lengths were cropped to exclude
 10px off each side, then normalized into 20 longitudinal bins. Data_Figure
 4C_ConstGFP_daughter transects_cropped.rds: Daughter cell data.
 Measurements taken at division. List of 104 elements, where each element
 is a matrix of fluorescence measurements across cell lengths. Each list
 element stands for a generation, and each matrix row is a cell measured at
 that generation. Columns are fluorescence levels at a given intracellular
 localization, from old pole to new pole. Lengths were cropped to exclude
 10px off each side, then normalized into 20 longitudinal bins. Data_Figure
 4C_ConstGFP_mother transects_cropped.rds: Daughter cell data. Measurements
 taken at division. List of 104 elements, where each element is a matrix of
 fluorescence measurements across cell lengths. Each list element stands
 for a generation, and each matrix row is a cell measured at that
 generation. Columns are fluorescence levels at a given intracellular
 localization, from old pole to new pole. Lengths were cropped to exclude
 10px off each side, then normalized into 20 longitudinal bins. Data_Figure
 4D_RpoS_daughter transects_cropped.rds: Daughter cell data. Measurements
 taken at division. List of 109 elements, where each element is a matrix of
 fluorescence measurements across cell lengths. Each list element stands
 for a generation, and each matrix row is a cell measured at that
 generation. Columns are fluorescence levels at a given intracellular
 localization, from old pole to new pole. Lengths were cropped to exclude
 10px off each side, then normalized into 20 longitudinal bins. Data_Figure
 4D_RpoS_mother transects_cropped.rds: Mother cell data. Measurements taken
 at division. List of 109 elements, where each element is a matrix of
 fluorescence measurements across cell lengths. Each list element stands
 for a generation, and each matrix row is a cell measured at that
 generation. Columns are fluorescence levels at a given intracellular
 localization, from old pole to new pole. Lengths were cropped to exclude
 10px off each side, then normalized into 20 longitudinal bins. Data_Figure
 5_RpoS_polar fluorescence.rds: List of 4 elements (1 = mother cells; 2 =
 daughter cells; 3 = daughter 2 cells; 4 = daughter 3 cells). Each element
 is a data frame, where each row is a measurement of a different
 generation. Data frames contain time (time.of.birth), old pole RpoS
 fluorescence at birth (op.bir), new pole fluorescence at birth (np.bir),
 old pole RpoS fluorescence at divison (op.div), new pole fluorescence at
 division (np.div), and a ratio op.bir/np.bir (bratio). Data_Figure
 5_ConstGFP_polar fluorescence.rds: List of 2 elements (1 = mother cells; 2
 = daughter cells). Each element is a data frame, where each row is a
 measurement of a different generation. Data frames contain time
 (time.of.birth), old pole fluorescence at birth (op.bir), new pole
 fluorescence at birth (np.bir), old pole fluorescence at divison (op.div),
 new pole fluorescence at division (np.div), and a ratio op.bir/np.bir
 (bratio). Data_Figure_6_old pole effect.rds: Data frame of mother cell
 data. Each row corresponds to a generation, and each column a measurement
 of cell physiology. It contains a cell lineage number (cell), length at
 birth (len.at.birth), mean fluorescence at birth (fluo1.at.birth), RpoS
 promoter activity (promoter), time (time.of.birth), and old pole length
 (oplen). The following measurements were recalculated by disregarding the
 old pole area of the cell: promoter2, fluo1.at.birth2, len.at.birth2.
 Paired daughter measurements are also provided for mean fluorescence
 (daughter.fluo1) and promoter activity (daughter.promoter). Data_Figure
 S1_RpoS lineage.rds: Sample cell lineage, containing all cells measured in
 a sample growth well. The data is a large list (2 elements) of raw DeLTA
 output converted to a working R format. The first element is a list in
 which each element is a cell, measured from birth until the cell is lost
 or dead. The second element is a large array containing the raw cell
 segmentation information. Data_Figure S1_RpoS mothers.rds: Data frame of
 mother cell data. Each row corresponds to a generation, and each column a
 measurement of cell physiology. It contains length at birth
 (len.at.birth), mean fluorescence at birth (fluo1.at.birth), RpoS promoter
 activity (promoter), and elongation rates (r). Data_Figure S1_Constitutive
 lineage.rds: Sample cell lineage, containing all cells measured in a
 sample growth well. The data is a large list (2 elements) of raw DeLTA
 output converted to a working R format. The first element is a list in
 which each element is a cell, measured from birth until the cell is lost
 or dead. The second element is a large array containing the raw cell
 segmentation information. Data_Figure S1_Constitutive mothers.rds: Data
 frame of mother cell data. Each row corresponds to a generation, and each
 column a measurement of cell physiology. It contains length at birth
 (len.at.birth), mean fluorescence at birth (fluo1.at.birth), constitutive
 promoter activity (promoter), and elongation rates (r). Data_Figure
 S2_RpoS stationary.rds: List of 2 elements, where the first contains
 mother cells and the second the daughters. Each element is a data frame of
 32,524 rows and 5 columns, containing paired data for every cell division.
 Each row shows data of one generation. Columns include time of birth in
 hours (time), time counted backwards from last maternal division
 (backwards.time), elongation rates (elong.rates), mean RpoS fluorescence
 at birth (fluorescence), and normalized fluorescence (norm.fluorescence).
 Data_Figure S3.rds: List of 2 elements (mother and daughter measurements),
 each a data frame of fluorescence measurements over generations.
 Data_Figure S6A_RpoS_mother transects.rds: Mother cell data. Measurements
 taken at birth. List of 109 elements, where each element is a matrix of
 fluorescence measurements across cell lengths. Each list element stands
 for a generation, and each matrix row is a cell measured at that
 generation. Columns are fluorescence levels at a given intracellular
 localization, from old pole to new pole. Lengths were normalized into 20
 longitudinal bins. Data_Figure S6A_ConstGFP_mother transects.rds: Mother
 cell data. Measurements taken at birth. List of 104 elements, where each
 element is a matrix of fluorescence measurements across cell lengths. Each
 list element stands for a generation, and each matrix row is a cell
 measured at that generation. Columns are fluorescence levels at a given
 intracellular localization, from old pole to new pole. Lengths were
 normalized into 20 longitudinal bins. Data_Figure S6B_RpoS_daughter
 transects.rds: Daughter cell data. Measurements taken at birth. List of
 109 elements, where each element is a matrix of fluorescence measurements
 across cell lengths. Each list element stands for a generation, and each
 matrix row is a cell measured at that generation. Columns are fluorescence
 levels at a given intracellular localization, from old pole to new pole.
 Lengths were normalized into 20 longitudinal bins. Data_Figure
 S6B_ConstGFP_daughter transects.rds: Daughter cell data. Measurements
 taken at birth. List of 104 elements, where each element is a matrix of
 fluorescence measurements across cell lengths. Each list element stands
 for a generation, and each matrix row is a cell measured at that
 generation. Columns are fluorescence levels at a given intracellular
 localization, from old pole to new pole. Lengths were normalized into 20
 longitudinal bins. Data_Figure S5_cellct_mat_per_frame.rds: Matrix
 representing the number of cells present in a given growth well over time.
 Rows indicate a growth well (1 to 244) and columns the movie frame, in 10
 min intervals (1 to 433). Data_Figure S5_endtrans_list_per_frame.rds: List
 of 433 elements, each containing a matrix of RpoS fluorescence transects.
 Each element of the list stands for a movie frame, in 10 min intervals. In
 the matrices, each row contains fluorescence measurements along the axis
 of the last cell in the growth well. Data_Figure
 S5_momtrans_list_per_frame.rds: List of 433 elements, each containing a
 matrix of RpoS fluorescence transects. Each element of the list stands for
 a movie frame, in 10 min intervals. In the matrices, each row contains
 fluorescence measurements along the axis of the mother cell. ##
 Sharing/Access information Links to other publicly accessible locations of
 the data: Proenca, A. M., Tuğrul, M., Nath A., & Steiner, U. K.
 (2023). Declining old pole physiology gradually enhances gene expression
 asymmetry in bacteria. BioRxiv.
 [https://doi.org/10.1101/2023.08.07.552301](https://doi.org/10.1101/2023.08.07.552301) ## Code/Software This submission contains the following R files, organized as to replicate the figures of the linked manuscript with the provided datasets: Figure 1.R Figure 2.R Figure 3.R Figure 4.R Figure 5.R Figure 6.R Figure S1.R Figure S2.R Figure S3.R Figure S5.R Figure S6.R Table S2.R Code was written in R v.4.1.2, using packages mgcv v.1.3-38, ggplot2 v.3.5.1, ggdensity v.1.0.0, and segmented v.1.4-0.