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.

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).

All image files can be opened using FIJI with Bioformats.