Data from: Competition for iron shapes metabolic antagonism between Bacillus subtilis and Pseudomonas

Description

Notes

Methods

Culturing

B. subtilis DK1042 (the naturally competent derivative of 3610) and Pseudomonas marginalis 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 MnCl₂ (LBGM), or King's B medium (KB; peptone 20 g/L, glycerol 1% (V/V), K₂HPO₄ 8.1 mM, MgSO₄·7H₂O 6.08 mM) at 30 °C. Colonies were grown on media solidified with 1.5% agar (W/V). FeCl₃, 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 OD₆₀₀ 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 OD₆₀₀ of 1.0 and the two strains were then mixed in equal volumes. For mixed B. subtilis cultures, wild type and mutant strains were mixed 1:1, 1:10, or 1:100, respectively, based on OD₆₀₀.

Stereomicroscopy

Interactions for stereomicroscopy were created using DK1042 carrying amyE::P_hyperspank-mKate2 and PS92 carrying attTn7::msfGFP. 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 Pseudomonas or Bacillus, respectively).