Menaquinone and iron are essential for complex colony development in Bacillus subtilis.

Cells of undomesticated species of Bacillus subtilis frequently form complex colonies during spreading on agar surfaces. Given that menaquinone is involved in another form of coordinated behavior, namely, sporulation, we looked for a possible role for menaquinone in complex colony development (CCD) in the B. subtilis strain NCIB 3610. Here we show that inhibition of menaquinone biosynthesis in B. subtilis indeed abolished its ability to develop complex colonies. Additionally some mutations of B. subtilis which confer defective CCD could be suppressed by menaquinone derivatives. Several such mutants mapped to the dhb operon encoding the genes responsible for the biosynthesis of the iron siderophore, bacillibactin. Our results demonstrate that both menaquinone and iron are essential for CCD in B. subtilis

Menaquinone derivatives lacking an isoprenoid side chain cannot suppress the complex colony development defect in <i>B. subtilis</i> siderophore mutants.

<p>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.</p

Menaquinone is essential for complex colony development and growth of <i>Bacillus subtilis</i>.

<p><b>A</b>. 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>B</b>. Growth curves of <i>B. subtilis</i> 3610 in spreading medium, and in spreading medium supplemented with 150 µM DPA. Each growth experiment was carried out in triplicate.</p

Growth curves of <i>B. subtilis</i> 3610 wild-type and <i>dhb</i> mutants.

<p>Growth curves were performed in spreading medium. The presented results are representative of three independent experiments.</p

The typical complex colony development phenotype of <i>B. subtilis</i> 3610.

<p>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.</p

Reconstitution of complex colony development by addition of MK-4.

<p>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.</p

Iron is essential for complex colony development in <i>Bacillus subtilis</i>.

<p><b>A</b>. 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>B</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 <i>dhb</i> 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.</p

Mutants in <i>dhb</i> genes are defective in bacillibactin (BB) synthesis.

<p><b>A</b>. 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>B</b>. Wild-type, <i>dhbE, dhbF</i> and <i>dhbA</i> 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. </p

Evolutionary dynamics of the Rap-Phr pathway.

<p>Rap-Phr expansion combines a horizontal mode of acquisition—systems diverge in separate lineages and then recombine by HGT—with a vertical mode of divergence—an autoinducer is duplicated and then one of the duplicates is modified. Social interactions govern both diversifying selection between lineages and acquisition of a diverged system [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000330#pbio.2000330.ref041" target="_blank">41</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000330#pbio.2000330.ref060" target="_blank">60</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2000330#pbio.2000330.ref061" target="_blank">61</a>]. Acquisition by horizontal transfer is sometimes followed by fixation in the lineage.</p