DOI: 10.5061/dryad.vq83bk3zc
title: Data from: Competition for iron shapes metabolic antagonism between Bacillus subtilis and Pseudomonas

description: Siderophores have long been implicated in sociomicrobiology as
 determinants of bacterial interrelations. For plant-associated genera like
 Bacillus and Pseudomonas, siderophores are often acclaimed for their
 function in biocontrol. Here, we set out to determine the functional role
 of the Bacillus subtilis siderophore bacillibactin in an antagonistic
 interaction with Pseudomonas marginalis. The presence of bacillibactin
 strongly influences the outcome of the interaction in an iron-dependent
 manner. A bacillibactin producer B. subtilis restricts the colony
 spreading of P. marginalis, repress the transcription of histidine
 kinase-encoding gene gacS, and thereby abolish production of secondary
 metabolites such as pyoverdine and viscosin. In contrast, the lack of
 bacillibactin restricts B. subtilis colony growth in a mechanism
 reminiscent of a siderophore tug-of-war for iron. Our study identifies a
 Bacillus-Pseudomonas interaction conserved across fluorescent Pseudomonas
 spp., expanding our understanding of the interplay between two genera of
 the most well-studied soil microbes.
description: Culturing <em>B. subtilis</em>
 DK1042 (the naturally competent derivative of 3610) and
 <em>Pseudomonas marginalis </em>PS92 were routinely cultured
 in lysogeny broth (LB, Lennox, Carl Roth, 10 g/L tryptone, 5 g/L yeast
 extract, and 5 g/L NaCl), LB supplemented with 1% (V/V) glycerol and 0.1
 mM MnCl<sub>2</sub> (LBGM), or King’s B medium (KB; peptone 20
 g/L, glycerol 1% (V/V),
 K<sub>2</sub>HPO<sub>4</sub> 8.1 mM,
 MgSO<sub>4</sub>·7H<sub>2</sub>O 6.08 mM) at 30
 °C. Colonies were grown on media solidified with 1.5% agar (W/V).
 FeCl<sub>3</sub>, ammonium ferric citrate and 2,2’-bipyridine
 (BPD) were supplemented into autoclaved agar at varying concentrations.
 Antibiotics were added in the following final concentrations: Gentamycin
 (Gm) 50 μg/mL, Ampicillin (Amp) 100 μg/mL, Chloramphenicol (Cm) 5 μg/mL,
 and Kanamycin (Km) 10 μg/mL. DK1042 and PS92 were
 routinely spotted at a 5.0 mm distance on agar surfaces using 2 µL culture
 at OD<sub>600</sub> of 1.0. Prior to spotting, plates were
 dried for 30 minutes in a lateral flow hood and were then incubated at 30
 °C. When spotting cocultures, each species was adjusted to
 OD<sub>600</sub> of 1.0 and the two strains were then mixed in
 equal volumes. For mixed <em>B. subtilis</em> cultures, wild
 type and mutant strains were mixed 1:1, 1:10, or 1:100, respectively,
 based on OD<sub>600</sub>.
 Stereomicroscopy Interactions for
 stereomicroscopy were created using DK1042 carrying
 <em>amy</em>E::P<em><sub>hyperspank</sub></em>-<em>mKate2</em> and PS92 carrying <em>attTn7::msfGFP</em>. Colonies were imaged with a Carl Zeiss Axio Zoom V16 stereomicroscope with a Zeiss CL 9000 LED light source and an AxioCam 503 monochromatic camera. The stereoscope was equipped with a PlanApo Z 0.5x/0.125 FWD 114 mm, and the filter sets 38 HE eGFP (ex: 470/40, em: 525/50) and 63 HE mRFP (ex: 572/25, em: 629/62). Exposure time was optimized for proper contrast. Timelapse series were acquired at 15-minute intervals for 72 hours. Bacterial cultures were spotted on agar solidified in a 35 mm petri dish which was then placed into a Tokai Hit stage top incubator pre-warmed to 30°C. Sterile MilliQ water was added to the incubator water bath to maintain constant humidity in the chamber. Image processing and analysis was performed in FIJI (2.1.0/1.53f51). Contrast in fluorescence channels was adjusted identically on a linear scale. Colony area was measured by segmenting the colony of interest with Otsu’s algorithm based on fluorescence (msfGFP or mKate2 for <em>Pseudomonas</em> or <em>Bacillus</em>, respectively).
description: All image files can be opened using FIJI with Bioformats.
description: # Title of dataset: Competition for iron shapes metabolic antagonism
 between Bacillus subtilis and Pseudomonas --- This dataset contains raw
 .czi images from the related manuscript "Competition for iron shapes
 metabolic antagonism between Bacillus subtilis and Pseudomonas" by
 Lyng et al. 2023. The study uses information about bacterial colony size
 to determine the cause behind a mutual antagonism between B. subtilis and
 P. marginalis. We routinely cultured colonies in close proximity on agar
 surfaces and imaged the colonies, using constitutively expressed
 fluorophores to identify and measure each strain. ## Description of the
 data and file structure The dataset is a collection of compressed files in
 .zip format. You will need compression software to unpack the .zip-files.
 Each .zip file contains images for an experiment from the associated
 manuscript. The images relate to the manuscript figures as such:
 "base_interaction" - Figure 1 and S1 "complementation"
 - Figure 2 and S4 "dhb_mutants" - Figure 2 and S4
 "iron_experiments" - Figure 3 "LBase_KBase" - Figure
 S2 "msgg_interaction" - Figure S2 "msi" - Figure 4 and
 S6 "mutants" - Figure 5 and S7 "other_ps" - Figure 6
 "timelapse" - Video S1 Images from non-supplementary figures
 have file names formatted according to the following structure:
 "medium_concentration_inoculationratio_interaction_strain.czi"
 When a variable is irrelevant to an image, it is populated with
 "NA". ## Sharing/Access information ImageJ Scripts for data
 acquisition and R scripts for figure reproduction can be found at
 ## Code/Software All
 images are in the Zeiss proprietary .czi format, which can be opened in
 FIJI () using BioFormats
 (). Microscopy montages
 were created by processing .czi files with the IJ script
 "colony_process.ijm" before using the plugin QuickFigures
 () to create the figure
 layout.