Figure 1.
The typical complex colony development phenotype of B. subtilis 3610.
Approximately 105 cells from the mid logarithmic growth stage were spotted on spreading plates containing 1.2% agar and incubated at 37°C as described in Methods. The plates were photographed at various time points (12, 60 and 72 hours) after inoculation. The presented results are representative of five independent experiments.
Figure 2.
Menaquinone is essential for complex colony development and growth of Bacillus subtilis.
A. Three μl of a fresh overnight culture of the wild-type strain were spotted onto the center of plates containing 1.2% agar supplemented with 150 µM DPA (left) or with 150 µM DPA + 100 µM MK-4 (right). Plates were photographed after 72 hours. Each experiment was carried out in triplicate. B. Growth curves of B. subtilis 3610 in spreading medium, and in spreading medium supplemented with 150 µM DPA. Each growth experiment was carried out in triplicate.
Figure 3.
The menG mutant is defective in complex colony development and growth.
A. Cells of wild-type (left) and menG (right) strains were spotted on spreading plates containing 1.2% agar and incubated at 37°C in a humid and dark environment. Plates were photographed after 72 hours. The presented results are representative of five independent experiments. B. Growth curves were performed in spreading medium or in spreading medium supplemented with 40 μg/ml of either MK-4 or phylloquinone. The presented results are representative of three independent experiments.
Figure 4.
Reconstitution of complex colony development by addition of MK-4.
Three μl of a fresh overnight culture were spotted onto the center of spreading plates containing 1.2% agar (left) and spreading plates containing 1.2% agar supplemented with 40 μg/ml MK-4 (right) and incubated at 37°C in a humid and dark environment. Plates were photographed after 72 hours. The presented results are representative of at least five independent experiments.
Figure 5.
Mutants in dhb genes are defective in bacillibactin (BB) synthesis.
A. Production of BB starts with chorismate and proceeds through the enzymatic activities of the DhbC, DhbB, and DhbA proteins to DHB, an intermediate with weak siderophore activity. DHB is subsequently activated by DhbE-mediated adenylation. A modular peptide synthetase later modifies the resulting 2, 3-dihydroxy-benzoyl-adenylate through the addition of glycine and threonine residues and finally esterifies three of these intermediates to form BB; B. Wild-type, dhbE, dhbF and dhbA strains were plated on CAS hard agar plates (Materials and Methods). Photographs were taken after 48 hours of incubation at room temperature. The results are representative of five independent experiments.
Figure 6.
Iron is essential for complex colony development in Bacillus subtilis.
A. Three μl of a fresh overnight grown culture were spotted onto the center of spreading plates containing 1.2% agar (left) and spreading plates containing 1.2% agar supplemented with 150 µM Fe+3 (right), and incubated at 37°C in a humid and dark environment. Plates were photographed after 72 hours. B. Three μl of a fresh overnight culture were spotted onto the center of spreading plates containing 1.2% agar (left) and spreading plates containing 1.2% agar supplemented with 120 μM of an iron chelator, 2,2'-dipyridyl (right) and incubated at 37°C in a humid and dark environment. Inhibition of complex colony development in the dhb mutants in the presence of 120 μM 2, 2'-dipyridyl was observed following 72 hours of incubation at 37°C. The presented results are representative of five independent experiments.
Figure 7.
Growth curves of B. subtilis 3610 wild-type and dhb mutants.
Growth curves were performed in spreading medium. The presented results are representative of three independent experiments.
Figure 8.
Menaquinone derivatives lacking an isoprenoid side chain cannot suppress the complex colony development defect in B. subtilis siderophore mutants.
Three μl of a fresh overnight culture were spotted onto the center of spreading plates containing 1.2% agar, spreading plates containing 1.2% agar supplemented with 40 μg/ml of either MK-4, phylloquinone, menadione sodium bisulfite (MBS) or Hydroquinone (HyQ), or spreading plates containing 1.2% agar supplemented with 10 μg/ml of menadione. Photos were taken after 72 hours. Each experiment was carried out in triplicate.