Population-level control of two manganese oxidases expands the niche for bacterial manganese biomineralization

https://doi.org/10.5061/dryad.fj6q5742z

Description of the data and file structure

All data were collected using the same microscope and the same settings, as described in the manuscript. The data is a compressed folder of all of the data used to produce the main figures and Supplementary figures, called "Gehin_Data_2025.zip". The folder is structured by the figure number, where the compiled raw data (collected after microcolony, cells, and aggregates segmentation were performed) can be found in a .csv or .mat format. In addition, the MATLAB scripts are provided as .m format. If no supplementary file is present, the data is directly contained within the first lines of the MATLAB script. 

Files and variables

File: Gehin_Data_2025_.zip

Description of data aquisition

Single cell segmentation. Scripts for single cells analysis and sample images are provided to extract the single cell from .tiff images. The parameters used for this study are the number of cells segmented and the average fluorescence per cell. This information was then compiled into .mat or .xlsx files and used to quantify the amount of fluorescence signal, number of cells fluorescing, and proportion of cells fluorescing.

Aggregate and microcolony segmentation. Scripts developed for aggregate identification are also provided, along with sample images. Those scripts also include the brightfield RGB processing for the identification and quantification of manganese oxides precipitates on the surface of aggregated cells or microcolonies. Phase contrast images were used for aggregates and microcolony segmentations, mCherry fluorescence channel was used for fluorescence extraction, and Brightfield for RGB color parameters.

Spatiotemporal analysis. Microcolony time-lapses were acquired at 30 min interval, capturing phase contrast and mCherry fluorescence sequentially, and specified in the "Time.xlsx" file. Scripts for time-lapse analysis are provided. Liquid time points sampling frequency is described in the .xlsx source files. 

ICP-MS. Aqueous manganese was measured using ICP-MS and the results are reported in .xlsx files, including the sample name, original dilution, dilution factors, amounts of acid added, ICP-MS reads, and calculation to concentrations in µM. When file is not provided, the ICP-MS data is contained in the .m scripts.

Plate reader. Growth curves of different strains were acquired using a Plate reader with 15 min time interval. The time and optical density (600 nm) are reported in individual .xlsx files. The fluorescence signal was collected and stored in separate files. 

False color images. The script and sample images are provided to generate false color of the segemented cells using the mask obtained during single cell segmentation analysis. 

Name convention:

• Bioreporter for mnxG promoter: PmnxG, P2447, P24
• Bioreporter for mcoA promoter: PmcoA, P2665, P26

* mnxG Gene deletion: Delta2447, Delta_mnxG
* mcoA Gene deletion: Delta2665, Delat_mcoA
* mnxG and mcoA double gene deletion: Delta2447_2665, DeltamnxGmcoA
* WT: for Wild-type
* t or T correspond to the time point, in hours. For microcolonies, the timepoints T correspond to the the time point from the beginning of imaging, with a time interval of 30 min.
* MST: acronym for the defined growth medium used in this study.

For microscopy images:
* M-Cherry is the m-Cherry fluorescence channel.
* PC corresponds to Phase Contrast channel.
* BrightfieldRed = Red channel in bright field imaging.
* 100x: 100x objective, resulting in 1000x effective magnification.

All fluorescence data has arbitrary units (a.u.) that are specific to the epifluorescence microscopy settings (described in the materials and methods of the manuscript). Manganese concentration data was collected in ppb and converted to µM. 

Missing values are indicated by NaN. 

Specific content:

Figure 1_MST with without Mn. This folder contains the original images used to extract fluorescence pixel intensity from the pictures of microcolonies of Pseudomonas putida GB-1. The name of the folder refers to the experimental conditions of the experiment. MST is the acronym for the defined growth medium that was used in the presence (Mn) or absence (_noMn) of 50 µM MnCl₂. The file contains a selection of images for the two strain used (P2447 and P2665). This file also contains a subfolder entitled "CROP", which contains cropped images of the image selection.

The codes "SegmentationPixelsP2447.m" and "SegmentationPixelsP2665.m" are provided to segment and extract the fluorescence intensity of microcolonies P2447 and P2665 strains. These MATLAB scripts quantifies colony-associated mCherry fluorescence from microscopy images across four experimental conditions (WT −Mn, PmnxG −Mn, WT +Mn, PmnxG +Mn). For each condition, all mCherry-channel images in a specified directory are processed to remove saturated pixels, estimate and subtract background fluorescence, and segment biomass-associated pixels using intensity thresholding. Fluorescence intensities are extracted exclusively from segmented biomass pixels and aggregated across images. Outliers are removed using a six–standard-deviation filter, and fluorescence distributions are compared across conditions using violin plots. A reference fluorescence threshold is defined as the 99.9th percentile of the WT +Mn condition and displayed for comparison. Statistical differences among conditions are evaluated using nonparametric Kruskal–Wallis tests followed by multiple-comparison analysis.

Figure 2_Spatiotemporal analysis. This file contains the data and codes used in the making of figure 2 and supplementary figures S5, S5, and S12. The folder "TimeLapseP2447" contains the time lapse information for the PmnxG bioreporter.  The folder "TimeLapseP2665" contains the time lapse information for the PmcoA bioreporter. Images used are provided in TIFF format. In each TimeLapse sub folder, TIFF images per time point can be found, as described in name convention section. Each P2447 and P2665 timelapse file also contains "ImageBinary.m" script used for the segmentation of the microcolonies, and "SpatioTemporal.m" code for extration of the flurescence signal in time and space.

"ImageBinary.m" and "ImageBinary2.m": This MATLAB script processes time-lapse mCherry fluorescence images to generate contrast-enhanced frames for visualization of temporal changes in colony fluorescence. Individual mCherry images acquired at successive time points are loaded, background fluorescence is estimated and subtracted using the median pixel intensity, and low-intensity pixels are removed using a fixed fluorescence threshold derived from a reference condition (specified in the script). The remaining fluorescence signal is contrast-scaled and rendered using a pseudocolor lookup table, and each processed frame is exported as a high-resolution image suitable for assembly into a time-lapse movie illustrating the spatial and temporal evolution of mCherry-expressing biomass.

"SpatioTemporal.m": This MATLAB script analyzes and visualizes the spatiotemporal distribution of mCherry fluorescence within microbial microcolonies using normalized coordinate data (XY) extracted from multiple time points. Fluorescent coordinates are filtered to retain only points within the microcolonies boundary, then combined to compare spatial organization across time. The script generates scatter plots, mean radial distributions, and microcolony envelopes to quantify shifts in fluorescence localization, enabling assessment of temporal changes in the internal structure and spatial dynamics of fluorescent subpopulations within microcolonies.

Each P2447 and P2665 timelapse subfolder contain time points subfolders corresponding to four separate time points. These time point are found in the name convention section (e.g., t1 contains _T47 = timepoint 47 of microcolony imaging with 30 min interval = 23.5 h time point). Each time point subfolder contains the selected TIFF images in phase contrast and m-cherry fluorescence channels. The description a, b, or c after the time point T corresponds to different images within the same time point. Additionally, a script named "ImageProcessingTemporal.m" can be found, which an associated matrix called "XY.mat". This matrix stores the normalized spatial coordinates of fluorescent cells within microcolonies. This MATLAB script identifies and analyzes fluorescence within individual microcolonies by selectively segmenting large mCherry-labeled microcolonies from time-lapse microscopy images. For each image, saturated pixels are removed, background fluorescence is estimated and subtracted independently for mCherry and phase-contrast channels, and aggregates are isolated using size-filtered binary segmentation. Fluorescent subregions within each aggregate are further thresholded to identify mCherry-expressing cells, which are spatially localized relative to the aggregate centroid and perimeter. Fluorescence coordinates are normalized by aggregate radius to enable comparison across colonies of different sizes, and the resulting normalized spatial distributions are aggregated and visualized to assess the radial organization of fluorescence within microcolonies.

Finally, the subfolder "TimeLapseP2665" contains an extra subfolder called "LateImages". This folder contains selected images form later time points (T70 to T82).

Figure 3 & SI 8_Pattern analysis. This file conains the data and codes for the analysis of the fluorescence pattern used in Figure 3 and supplementary Figure S8. Data consist of cross-section intensities (intensity profiles) of the microcolonies of the PmnxG and PmcoA bioreporters. The code "PatternAnalysis.m" provided is used to normalize the intensity profiles and averages them between replicates. The representative images are also provided in this file. Contrasts and fluorescence intensities were normalized and the fluorescence was falsed colored using heatmap. The folder is organized in two subfolders: P2447 and P2665 for each strain studied. In each folder, the moving average cross-section are available under the name "movval_X.mat", with X the time point number corresponding to the time point of the selected images. For the exact time point, consult the provided script "PatternAnalysis.m", also provided in the subfolder.

"PatternAnalysis.m": This MATLAB script performs post-processing spatial pattern analysis of time-lapse mCherry fluorescence in P2447 by quantifying how intensity varies across the normalized colony radius (“relative diameter”) over time and across replicates. Radial fluorescence profiles (movval_*.mat) from six replicates are loaded, rescaled to a common 0–1 spatial axis, binned to a shared grid, and averaged to generate a mean spatiotemporal fluorescence profile. The script visualizes individual and replicate-averaged radial profiles using time-coded colormaps, fits smooth high-order polynomials to selected time points, and exports publication-ready figures summarizing temporal changes in fluorescence localization patterns (e.g., center vs edge enrichment) across the colony.

Figure 4_Fluorescence image. This file contains the original images as .tiff before and after contrast adjustments. "ImageBinary.m" codes are provided for segmentation and analysis of the fluorescence intensity and heatmaps false coloring. Images containing "Crop_" label are cropped copies of the provided TIFF images.

"ImageBinary.m": This MATLAB script generates mCherry fluorescence snapshots from multiple colony and liquid-culture images (P2447 and P2665) by applying consistent background subtraction and display scaling. For each selected image, a constant background offset is subtracted (user-defined per dataset), negative/low-signal pixels are zeroed, and the resulting fluorescence is rendered with a pseudocolor colormap and standardized intensity limits. Processed frames are exported as high-resolution JPEGs for figure panels and/or assembly into movies. The script also includes an experimental (optional) step to estimate colony-centered radial intensity profiles by segmenting the colony, identifying its centroid and radius, and sampling fluorescence along multiple radial transects.

Figure 4_Mn Oxide vs Fluo kinetics. This file contains the data as .mat files and codes to segment and analyse the fluorescence and Mn oxide presence on microcolonies. This folder contains MATLAB scripts "ColorFluoAnalysis.m" and "ColorFluoAnalysisWT.m", and processed outputs used to quantify microcolony fluorescence (mCherry) and manganese oxide (MnOx) formation from multi-channel time-lapse microscopy (phase contrast, RGB brightfield, and mCherry). The analysis segments microcolonies from fluorescence images, extracts region-based metrics (mean intensity, percent colony area above fluorescence or MnOx thresholds), quantifies spatial overlap between fluorescence-positive and MnOx-positive regions, aggregates replicate microscope positions by strain, and generates time-resolved kinetic plots with mean ± variability.

• "mCherry.mat" – Per-position time series of mean mCherry fluorescence intensity within microcolonies.
• "MnOx.mat" – Per-position time series of mean MnOx proxy intensity derived from RGB brightfield images.
• "PropMCH.mat" – Percentage of colony area expressing mCherry over time.
• "PropMn.mat" – Percentage of colony area covered by MnOx over time.
• "PropOvl24.mat" – Percentage overlap between fluorescence-positive and MnOx-positive regions within colonies.
• "GroupF24.mat", "GroupMn24.mat" – Grouped matrices used for cross-condition statistical comparisons.
• "AvDark_7.mat", "AvDark_28.mat" – Colony-averaged MnOx intensity time series for defined analysis batches.
• "AvFluoInt_7.mat", "AvFluoInt_28.mat" – Colony-averaged fluorescence intensity time series for the same batches.
• "ssP24.mat", "ssP26.mat" – Microcolony size distributions extracted from segmentation.
• "ssProp24.mat", "ssProp26.mat" – Steady-state distributions of colony-level fluorescence coverage.

Figure SI 3_Liquid washed Mn no Mn. The file contains sample images of liquid cultures of Pseudomonas putida GB-1 wild-type (WT) and bioreporters. The .mat files provide the extracted fluorescence intensity per segmentated cell. The codes "SegmentationPixels.m" and "WashStat_MnnoMn.m" were used to average the replicates and plot the multimodal fluorescence signal as violin plots.

"SegmentationPixels.m": This MATLAB script segments bacterial microcolonies based on autofluorescence (GFP/mCherry channel) and quantifies single-pixel fluorescence intensity distributions across multiple experimental conditions. Images are loaded from predefined experiment directories corresponding to wild-type and mutant strains grown with or without Mn(II). For each condition, fluorescence images are background-corrected using the 3rd percentile intensity, concatenated across all time points, and reshaped into a single intensity vector representing colony-associated pixels.

The script compares fluorescence intensity distributions between conditions using violin plots, histograms, and non-parametric statistics (Kolmogorov–Smirnov tests and Kruskal–Wallis ANOVA with post hoc comparisons). High-intensity pixel populations are isolated to assess shifts in expression levels under Mn exposure. Summary figures are exported for downstream visualization and statistical interpretation.

"WashStat_MnnoMn.m": This MATLAB script analyzes single-cell–scale fluorescence distributions to test whether manganese (Mn) is required to maintain reporter expression during stationary phase. It uses previously segmented fluorescence pixel datasets extracted from washed cultures of wild-type and mutant strains (P2447 and P2665) grown with or without Mn(II). For each condition, pixel-level fluorescence intensities from multiple replicate images are concatenated, baseline-corrected, and filtered to remove extreme outliers. The script compares fluorescence intensity distributions across genotypes and Mn conditions using violin plots and non-parametric statistics (Kolmogorov–Smirnov, Kruskal–Wallis, and ANOVA). Separate analyses are performed for Mn-free and Mn-amended conditions, enabling direct assessment of Mn dependence for maintaining fluorescence after washing. Publication-ready violin plots are generated for both strains and conditions.

• "Composite_WT_noMn.tif" – Composite fluorescence image of wild-type cells washed and incubated without Mn.
• "CompositeP2447_noMn.tif" – Composite fluorescence image of P2447 cells washed and incubated without Mn.
• "CompositeP2665_noMn.tif" – Composite fluorescence image of P2665 cells washed and incubated without Mn.
• "PixFluo_WTnoMn.mat" – Fluorescence pixel intensities from washed wild-type cells without Mn.
• "PixFluo_WTMn10.mat" – Fluorescence pixel intensities from washed wild-type cells with 10 µM Mn.
• "PixFluo_WTMn50.mat" – Fluorescence pixel intensities from washed wild-type cells with 50 µM Mn.
• "PixFluo_noMn.mat" – Fluorescence pixel intensities from washed P2447 cells without Mn.
• "PixFluo_Mn10.mat" – Fluorescence pixel intensities from washed P2447 cells with 10 µM Mn.
• "PixFluo_Mn50.mat" – Fluorescence pixel intensities from washed P2447 cells with 50 µM Mn.
• "PixFluo_P2665_noMn.mat" – Fluorescence pixel intensities from washed P2665 cells without Mn.
• "PixFluo_P2665_Mn50.mat" – Fluorescence pixel intensities from washed P2665 cells with 50 µM Mn.

Figure SI 5_Liquid timelapse. This files contains the growth of the bioreporters and WT using optical density (OD) .csv in arbitrary absorbance units. "OD_P24Cl3.csv" contains the OD at each time point for six biological replicates. The CL3 corresponds to clone 3, the specific strain used here. The letter and number in parenthesis is the well coordinated from the 96-well plate used in this experiment. Similarly, "OD_P26Cl1.csv" corresponds to the growth measurements for the strain P2665, specified as clone no 1. 

Files "P2447.csv" and "P2665.csv" contain the average proportion of population ("prop P2447" or "prop P2665") expressing the reporter genes for each time point (in hour), expressed as a fraction. This table also contain the standard deviations for the proportion of population fluorescing ("std prop"), the normalized average fluorescence intensity ("fluo norm") in arbitrary units, and the standard deviation for the nomalized average fluorescence intensity ("std fluo norm").

Contact the corresponding author for the raw data. The "Time.csv" file contains the time serie of the OD measurements in seconds, matching the OD measurements in files "OD_P24Cl3.csv" and "OD_P26Cl1.csv".

Data processing and plotting was done using "GrowthFluo.m": This script combines growth and fluorescence measurements to compare wild-type and mutant strains across time. It imports optical density (OD) data from plate-reader assays and fluorescence data from single cells and aggregates, normalizes both to their respective maxima, and aligns growth and expression timelines. The script computes averages, standard deviations, and linear growth fits, then generates dual-axis plots showing biomass accumulation alongside promoter activity and fluorescence intensity for each strain. Output figures summarize growth–expression coupling for P2447 (PmnxG) and P2665 (PmcoA) clones under stationary-phase conditions.

Figure SI 6_Biofilm covered area. This file contains the codes to process the biofilm present in liquid cultures and extract the projected surface coved by cells with active bioreporters (shown by fluorescence signal above the threshold described in the associated manuscript).

"BiofilmAnalysisP2447.m": This script segments microcolonies from phase-contrast images and quantifies mCherry fluorescence within large aggregates. It applies background subtraction, intensity rescaling, Gaussian filtering, and global thresholding to identify colonies, then filters objects by size to retain aggregates only. Fluorescence pixels above a defined threshold are classified within segmented colonies, and the proportion of each aggregate surface expressing fluorescence is calculated. The script also generates false-color overlay images highlighting fluorescing regions within colonies and exports high-resolution figures for visualization.

"BiofilmAnalysisP2665_WT.m": This script segments large microcolonies from phase-contrast images and quantifies mCherry fluorescence within those aggregates for the P2665 strain. It performs background subtraction, intensity rescaling, Gaussian smoothing, and global thresholding to identify colonies, then filters objects by size to retain aggregates only. Fluorescent pixels above a defined threshold are classified within each colony, false-color overlays are generated to visualize fluorescing regions, and the percentage of colony area expressing fluorescence is calculated and exported for downstream analysis.

Figure SI 7_Photobleaching. This file contains the change in flurescence intensity of a microcolony as a function of exposure time. This shows the photobleaching of the fluorophores and contains the data as .mat files and codes as for processing of that data as .m file. 

"PostPhotobleaching.m":This script analyzes photobleaching as a function of repeated LED excitation pulses. It loads four replicate datasets containing percent photobleaching values, plots each replicate individually, and then computes the mean and standard deviation across replicates. The averaged photobleaching curve is displayed with error bars as a function of pulse number, and a representative quadratic fit with R² is annotated for reference. The output figures summarize bleaching behavior under repeated 450 nm LED exposure (1.7 W, 500 ms pulses).

Files in this folder

• "Perc1.mat" — photobleaching percentages for replicate A
• "Perc2.mat" — photobleaching percentages for replicate B
• "Perc3.mat" — photobleaching percentages for replicate C
• "Perc4.mat" — photobleaching percentages for replicate D

Figure SI 9_Livelyness. This file contains the .m code to process the lag phase and exponential phase data as a proxy for the livelyness of the cells. The data is embedded in the code. 

"LivelynessPostProcess.m": This script analyzes single-cell growth metrics to compare reporting and non-reporting populations for P2447 (PmnxG) and P2665 (PmcoA). It computes mean lag time and division time from manually curated datasets, visualizes group averages with overlaid individual measurements, and performs statistical comparisons using one-way ANOVA with post-hoc tests. Final outputs include bar plots, box plots, and summary figures used to quantify differences in growth dynamics between strains and expression states.

Figure SI 10_OpenChamberColonySize. The file contains the data and code to measure and plot the distribution of colony sizes across all experiments. This file contains:

"OpenChamberColonySize.csv": A table containing the colony number, the diameter in pixel (px), the diameter in microns (um), and the surface area in squared microns (um2).

"SurfaceChamber.csv": a list of all the surface areas of the microcolonies segmented in squared microns.

"ColonySizeDistribution.m": This script compares microcolony surface-area distributions between open and sealed chamber growth conditions. Colony size measurements are imported from spreadsheet files, grouped by chamber type, and visualized using violin plots to highlight differences in size distributions. The output figure summarizes how chamber geometry influences colony surface area.

Figure SI 11 & 15_ColonyOpenChamber profiles. This folder and subfolder contains sample images and data of macrocolonies grown on MST-agarose surfaces. The amount of Mn oxides were quantified using RGB image subtraction as described in the main text and the fluorescence intensity of the bioreporters. The pattern of fluorescence was assessed using the provided "ColonyProfileProcessing.m" code.

Images are cropped sections of original images, provided as both .jpg and .tif formats. This set of images were acquired with a 4x objective, resulting in 40x magnification. Color channels (RGB) were added to generate and RGB color image. Images with "_Subtracted" labels are original images with red channel subtracted to enhance the detection of Mn oxides.

The subfolder "Fluorescence processed" contains the original TIFF images of the m-cherry channel and the script to generate the processed images. The script "ImageBinary.m" processes cropped mCherry fluorescence microscopy images from colony and liquid culture experiments. Images are background-corrected using median intensity subtraction, thresholded to remove low-signal pixels, and displayed using a consistent false-color scale. The script exports high-resolution, images as .png with standardized colorbars to enable direct visual comparison of fluorescence intensity across strains, time points, and growth conditions.

The subfolder "Intensity Profiles" contains the cross-sections m-cherry fluorescence profiles. This subfolder includes "MnOx_t46.csv" table showing the intensity values for each pixel of the profiles. These intensity values (in relative units) show the intensity of the Mn oxide presence along the cross-section for the time point 46 (or 23h), and for 10 replicates (column B-K). The first column (A) corresponds to the relative cross-section index. The files "P2447_t46.csv" and "P2665_t46.csv" show the fluorescence intensities for 5 and 6 replicates, respectively. The columns are the replicates and the lines are the corresponding raw fluorescence intensity for each cross-section index. The processing script "ColonyProfileProcessing_T46.m" processes radial fluorescence intensity profiles extracted from bacterial colonies and Mn oxide images at a single time point (t46). Intensity profiles are normalized by colony diameter, resampled onto a common relative diameter scale, and averaged across colonies to generate representative radial profiles. The output is an averaged, normalized radial intensity profiles with confidence intervals for reporter strains and Mn oxides.

The subfolder "Knockout colonies" contains two following subfolders: "del2447" and "del2665", corresponding to the single knockout strains, lacking the genes 2447 or 2665, respectively. Each of these file contains the color combined (RGB stacked) images of colonies as .jpg and .tif formats. The "subtracted" means that the red channel has been subtracted to the gray images to enhance Mn oxide detection. All images were cropped from the original images to isolate single colonies. Finally, the subfolder "Intensity Profile" is also contained to process the knockout images. This file contains "IntProf_delP2447.csv" and "IntProf_del2665.csv". These files contain the Mn oxide absorbance intensity (a.u.) for each replicate (in column) for either delta-2447, or delta-2665 strains, respectively. The provided processing script called "ColonyProfileProcessing_T46.m" processes radial fluorescence intensity profiles extracted from individual colonies to compare ΔmnxG, ΔmcoA, and wild-type Mn oxide distributions. Pre-extracted intensity profiles from Excel files are used to compute colony diameters, normalize profiles by relative radius, and average profiles across colonies of different sizes. Mean intensity profiles and variability (standard deviation) are calculated for each condition and visualized as averaged radial profiles. The script generates a summary figure showing spatial intensity distributions as a function of normalized colony diameter and saves the output as "IntensityProfileDel_colony.svg".

The subfolder "Whole colonies" contains all the Mn oxide intensity profiles (MnOx_) at time points 46h, 58h, and 72h. Each column corresponds to a replicate. Similarly, "P2447_t...csv" and "P2665_t....csv" report the bioreporter m-cherry signal intensity across a profile for the time points 46h, 58h, 72h, and 101h. Each column correspond to a replicate. The script "ColonyProfileProcessing.m" analyzes radial fluorescence and Mn oxide intensity profiles of bacterial colonies across multiple time points (46, 58, 72, and 101 h). Pre-extracted intensity profiles from Excel files are used to estimate colony diameters, normalize intensity values by relative radial position, and average profiles across colonies exceeding a minimum size threshold. Mean radial profiles and 95% confidence intervals are calculated for each strain and time point. The script generates comparative figures showing temporal evolution of reporter fluorescence (PmnxG and PmcoA) and Mn oxide accumulation as a function of normalized colony radius, and saves the resulting plots as SVG files.

Figure SI 14_MicrocolonyOringineMnOx. This folder contains the .tiff and .png images with normalized fluorescence images and RBG color subtracted images to cross-reference the location of the fluorescence signal and the manganese oxides precipitates. Each image is duplicated into TIFF and PNG.

Figure SI 20_Biofilm analyis. This folder contains the data for the analysis of biofilms (or aggregated cells) in liquid growth medium. The color images in TIFF and PNG formats are provided in the subfolder "P2447" and "P2665" inside of the "Biofilm bioreporter MnOx 18h" folder. This folder also contains the "CropInfo" text file, which described the cropping dimensions used. The following processing scripts are also provided:

"BiofilmAnalysisKinetics50.m": This script processes phase-contrast and fluorescence microscopy images to segment bacterial microcolonies and quantify reporter fluorescence at the colony scale. Images are background-corrected and contrast-adjusted, followed by Gaussian filtering and threshold-based segmentation to identify colonies. Fluorescence intensity is analyzed within segmented colonies to classify pixels above a defined threshold, generate a false-color fluorescence map, and calculate both the fraction of fluorescent pixels and mean colony fluorescence intensity. The script outputs a false-color image ("FalseColorFluo.png") and summary metrics describing population-level fluorescence.

"BiofilmAnalysisP2447.m": This MATLAB script batch-processes paired phase-contrast (PC) and mCherry fluorescence .tif images from a specified experiment directory. It automatically filters the folder listing, loads the PC and fluorescence frames into memory, then segments microcolonies from the fluorescence channel using contrast scaling, Gaussian smoothing, Otsu thresholding, hole filling, and light morphological cleanup (erode/dilate). Large objects (area threshold) are retained as colony masks. For each segmented colony, the script performs background correction and applies a fixed fluorescence threshold to classify pixels as “fluorescent” vs “non-fluorescent,” generates a false-color overlay image (FalseColorFluo.png), and reports percent fluorescent coverage per colony (PercFluo) based on the fraction of above-threshold pixels within each colony region.

"BiofilmAnalysisP2665_WT.m": This script batch-processes phase-contrast (PC) and mCherry fluorescence .tif images from the P2665, 50 µM Mn(II) dataset to (1) segment microcolonies and (2) quantify the fraction of each colony area that is fluorescent. It scans the working folder for matching PC.tif and herry.tif files, imports them into memory, then for a selected frame (e = 3) performs colony segmentation by intensity rescaling, Gaussian smoothing, Otsu thresholding (imbinarize), hole-filling, erosion, and a large dilation to merge colony regions. Colonies are filtered by area (Area > 16900 px) and fluorescence is background-corrected (median subtraction) and thresholded (Thresh = 700) to identify “on” pixels within colony masks. The script generates and exports a false-color mask image (FalseColorFluo.png, background/colony/fluorescent-colony pixels coded as 0/1/2) and computes percent fluorescent coverage per colony (PercFluo, based on fluorescent pixels divided by colony area).

"BiofilmKinetics.m": Combines replicate biofilm fluorescence metrics (percent aggregates fluorescing and/or normalized fluorescence) with Mn oxide precipitation time series to compute NaN-aware means ± error bands and export dual-axis composite kinetics plots (raw time and stationary-phase-shifted).

Code/software

MATLAB version 2021a