Abstract
Non-photosynthetic bacteria often respond to changes in light. These responses are usually regulated by photoreceptor proteins, but the mechanism of light response in biofilms is poorly understood. Here, we show that colony biofilms of Bacillus subtilis display light responses that are not dependent on typical photoreceptor proteins. Under light, B. subtilis biofilms do not mature and instead keep on expanding, resulting in thin, smooth colonies with low pigmentation. Similar effects have been previously observed upon inhibition of the biosynthesis or export of pulcherriminic acid, an iron chelator known to inhibit colony expansion by reducing extracellular iron levels. We show that light induces spontaneous degradation of pulcherriminic acid. In addition, Fe3+-bound pulcherriminic acid upregulates the yvmC operon (which is responsible for pulcherriminic acid biosynthesis) by inhibiting the binding of a repressor protein (PchR) to the yvmC promoter. Thus, the photosensitivity of pulcherriminic acid enables this metabolite to control iron availability, yvmC expression, and biofilm development in response to changes in light conditions.
Similar content being viewed by others
Introduction
Light exerts a profound influence on the physiology and behavior of almost all living organisms, including non-photosynthetic bacteria. Like other organisms, many non-photosynthetic bacteria respond to light using photoreceptor proteins1,2,3. These proteins are composed of chromophore-bearing photosensor domains and a range of output domains, including histidine kinase, GGDEF, EAL, and DNA-binding domains3. This allows the photoreceptor proteins to transduce light signals into transcriptional responses. Photoreceptor proteins regulate diverse functions including motility, biofilm formation, infection, and biosynthesis2,4,5,6,7,8,9,10. For instance, many non-photosynthetic bacteria, including soil bacteria, Myxococcus xanthus, Streptomyces coelicolor, and Bacillus megaterium, synthesize protective pigments, termed carotenoids, in response to light to counteract photooxidants11,12. The enterobacterium Escherichia coli employs light and low temperature as cues to discern that it is outside its host, inducing biofilm formation and repressing adhesive curli fimbriae13. The leaf-associated bacterium Pseudomonas syringae employs light as an anticipatory cue for subsequent water loss, inducing genes for water stress adaptation14. These reports indicate that the light response of non-photosynthetic bacteria may vary among species depending on their lifestyles and habitats.
In their natural habitat, most bacteria live in sessile multicellular communities, termed biofilms. In biofilms, bacteria are embedded in a self-produced matrix of extracellular polymeric substances (EPS), comprising exopolysaccharides, protein polymers, and DNA15. This matrix protects resident bacteria from antibiotics, the host immune response, and environmental challenges15. Accordingly, the biofilm mode of growth is regarded as an adaptive strategy to the natural environment, distinct from the planktonic mode. It is therefore of interest to test whether photoreceptors may play a specific role in biofilms. However, while many photoreceptors have been identified that regulate gene expression in planktonic cells or control the lifestyle choice between planktonic and biofilm growth modes4, their function in biofilms remains largely unstudied.
We happened to find that light exposure altered the biofilm morphology of the Gram-positive spore-forming bacterium Bacillus subtilis. B. subtilis is capable of forming robust biofilms, such as colony biofilms and floating biofilms, which are dependent on EPS components, specifically exopolysaccharides and protein polymers of TasA and BslA16,17. In the environment, B. subtilis biofilms thrive in soil and plant rhizosphere, so they can reach the soil surface and be exposed to dangerous sunlight. To avoid this situation, B. subtilis biofilms may sense light, but their light-sensing mechanism is poorly understood. B. subtilis has one possible photoreceptor, the light-oxygen-voltage (LOV) domain protein YtvA18, which is a component of the signal transduction pathway for the stress-responsive alternative sigma factor, σB 19. However, while blue light exposure induced a series of conformational changes in YtvA in vitro18, the exposure did not induce expression of σB-regulated genes in the wild-type strain20,21. This suggests that YtvA plays only a minor role or functions under unknown conditions in vivo. Recently, B. subtilis was reported to possess a circadian system22,23. In response to dark/light cycles, B. subtilis biofilms exhibited oscillations in ytvA and kinC transcription, which persisted after the biofilms were transferred to constant dark or light conditions22,23. While the mechanism of this circadian system remains unknown, the oscillations in ytvA and kinC transcription did not require YtvA23. These observations suggest that B. subtilis biofilms may possess a light-sensing mechanism that is not dependent on typical photoreceptor proteins.
Here, we investigated the light response of B. subtilis biofilms. Under a growth-permissive level of light, the biofilms did not mature and continued to grow. This phenotype was attributed to a reduction in the iron chelator, pulcherriminic acid. Upon exposure to light, purified pulcherriminic acid was rapidly degraded, indicating that its photosensitive property allows pulcherriminic acid to control the light response of biofilms in response to changes in light conditions. These findings reveal the unusual involvement of this molecule in the light response of B. subtilis biofilms.
Results
Light stress response in B. subtilis
Visible light serves as an environmental cue, but excess light induces a stress response in bacteria. To avoid confusing the light response and the light stress response in biofilm analysis, we first tried to elucidate the light stress response in the B. subtilis strain NCIB 3610 [hereafter referred to as the wild-type (WT) strain]16 under non-biofilm conditions. For this purpose, serial dilutions of an overnight culture of the WT strain were grown in LB solid medium under dark and light conditions (Fig. 1). In LB medium, B. subtilis exhibits rapid growth but does not form biofilms. Continuous exposure to intense light (110 μmol m−2 s−1, which is approximately equivalent to the brightness on a cloudy day) from fluorescent lamps prevented colony formation in low-density inoculation, whereas moderate light exposure (35 μmol m−2 s−1) had little or no effect on colony formation. The observed growth suppression was due to the cumulative effect of light exposure rather than the immediate effect, as 3 h of intense light exposure did not prevent colony formation. No growth suppression was observed in LB medium supplemented with ROS scavengers, cysteine and ascorbic acid. These observations suggest that intense light exposure may stimulate the endogenous generation of ROS, thereby preventing colony formation in low-density inoculation.
Overnight cultures of the WT strain were serially diluted, and 2 μL of the dilutions were spotted on LB agar medium. Plates were incubated at 30 °C for 24 h under dark or indicated light conditions. Light intensities: intense light, ~110 μmol m−2s−1; moderate light, ~35 μmol m−2s−1. Cysteine and sodium L-ascorbate were added at 1 mM (final). These experiments were performed three times with similar results.
To identify the cause of the light sensitivity, we isolated transposon insertion mutants that were resistant to intense light. All isolated light-resistant mutants had transposon insertions within the hemAXCDBL operon (Fig. 2a, b), which encodes enzymes for heme biosynthesis24. Specifically, three transposons were inserted within the hemA promoter. The impact of these insertions on the expression of the hemA operon was evaluated using the PhemA-gfp transcriptional reporter. Flow cytometry revealed that the PhemA-gfp reporter produced strong GFP fluorescence under both dark and intense light conditions (Supplementary Fig. 1). The transposon insertions within the hemA promoter resulted in a significant reduction of its expression, rather than a complete abolition. Given that the transposon insertions disrupted the hemA promoter, the observed weak expression is probably induced by a promoter within the transposons and supports a minimal level of heme synthesis required for normal growth24. The other eight transposons were inserted immediately upstream of or within hemL. B. subtilis possesses a hemL paralog, gsaB, which is probably the reason why so many intragenic transposon insertions were observed in hemL, despite the essentiality of heme biosynthesis for normal growth24. The deletion of hemL resulted in light resistance, confirming that the transposon insertions in hemL were responsible for the light resistance (Fig. 2b). These findings suggest that the observed light resistance is attributable to decreased heme biosynthesis.
a Transposon (Tn) insertion site in the hemA operon. Tn insertion sites relative to the start site of hemA (1368 bp) or hemL (1290 bp) are shown in brackets. The putative -10 and −35 regions of the hemA promoter are indicated by red letters. Tn mutants used in b are indicated by red letters. b Colony growth of representative light-resistant Tn mutants and the ΔhemL mutant. Serial dilutions of the indicated strains were grown under dark and intense light conditions. c Iron supplementation suppresses light sensitivity. Serial dilutions of the WT strain were grown on LB medium with or without 150 μM FeCl3. d Schematic representation of the heme biosynthetic pathway. Protoporphyrin IX and related enzymes are indicated by yellow-highlighted and red letters, respectively. e The structure of Pspac-hemHY and Pspac-hemY strains. In these strains, the pMutin vector containing an IPTG-inducible spac promoter and lacI, is inserted upstream of hemH or hemY, thereby placing hemHY or hemY under the control of the spac promoter. f Increased expression of hemHY and limited expression of hemY result in light resistance. Serial dilutions of the indicated strains were grown in LB medium supplemented with 0 to 100 μM IPTG. Colony assays were performed three times with similar results.
The addition of 150 μM FeCl3 to LB medium completely suppressed light sensitivity (Fig. 2c). To test whether iron supplementation might affect expression of the hemA operon, expression of the PhemA-gfp reporter was examined in LB and LB + 150 μM FeCl3 under both dark and intense light conditions. Flow cytometry revealed that iron supplementation resulted in a modest reduction in PhemA-gfp expression, particularly under intense light conditions (Supplementary Fig. 2a). To quantify the changes in PhemA-gfp expression, crude lysates were extracted from colonies and analyzed for GFP levels by SDS-PAGE. SDS was previously reported to have a negligible effect on GFP fluorescence under the weak alkaline conditions, typically used in SDS-PAGE analysis25. Quantitative analysis demonstrated that iron supplementation reduced PhemA-gfp expression by threefold under intense light conditions (Supplementary Fig. 2b, c). Given that LB medium contains only trace amounts of iron (5.3 ± 0.4 μM)26, these observations indicate that iron deficiency in LB medium may lead to increased hemA operon expression, thereby increasing light sensitivity.
Blue light has been demonstrated to excite protoporphyrin IX, an intermediate in heme biosynthesis, and generate ROS (Fig. 2d)12,27. Given that both decreased expression of the hemA operon and disruption of hemL reduce protoporphyrin IX levels, we predicted that this ROS generation mechanism would be responsible for the light sensitivity. To evaluate the prediction, we examined whether changes in protoporphyrin IX levels influence light sensitivity. In B. subtilis, protoporphyrin IX is produced from protoporphyrinogen IX by HemY and is used by HemH to produce iron-containing heme B (Fig. 2d). To modulate protoporphyrin IX levels, the IPTG-inducible spac promoter28 was inserted upstream of hemH or hemY within the hemEHY operon in the chromosome (Fig. 2e). In the resulting Pspac-hemHY and Pspac-hemY strains, strong expression of hemHY with 100 μM IPTG and limited expression of hemY with 10 μM IPTG, both of which were expected to reduce protoporphyrin IX levels, resulted in light resistance (Fig. 2f). These results indicate that intense light exposure stimulates protoporphyrin IX-dependent ROS generation, thereby increasing light sensitivity, particularly under iron-deficient conditions.
We determined whether light exposure had a comparable effect on the growth of the WT strain under biofilm-inducing conditions. As observed in LB medium, intense light prevented colony formation in MSgg medium, a widely used condition for inducing biofilm development16 (Supplementary Fig. 3a). This light sensitivity was also the result of the protoporphyrin IX-dependent mechanism, as it was suppressed by transposon insertions in the hemA operon and supplementation with cysteine, ascorbic acid, or iron (Supplementary Fig. 3a, b). Furthermore, moderate light did not affect colony formation in MSgg medium. Consequently, in subsequent biofilm analyses, moderate light was used as the standard light condition, which permitted normal growth without markedly stimulating protoporphyrin IX-dependent ROS generation. Should such stimulation persist under moderate light, its effect can be suppressed by ROS scavengers.
Light induces biofilm expansion
We investigated whether moderate light affects the growth and development of colony biofilms. To this end, 100-fold dilutions of an overnight culture of the WT strain were grown in MSgg medium under dark and light conditions. In the dark, colony biofilms almost stopped expanding at 72 h and exhibited thickening and wrinkling of their surfaces by 120 h (Fig. 3a). By contrast, under moderate light (35 μmol m−2s−1), colony biofilms continued to expand for at least 120 h and produced a thin and smooth morphology. The average area of colony biofilms at 120 h was 1.9 and 9.3 cm2 under dark and moderate light conditions, respectively (Fig. 3b). In a plate with one half covered with aluminum foil, moderate light promoted colony expansion in the bright area but not in the shaded area (Fig. 3c). These observations demonstrate that B subtilis biofilms are responsive to a growth-permissive level of visible light. To determine the minimal light intensity and specific wavelength that induce colony expansion, biofilm growth was further examined at lower light intensities and different wavelengths. Low light (14.5 μmol m−2s−1) promoted biofilm expansion, whereas dim light (4.8 μmol m−2s−1) did not (Fig. 3d). Blue LED light (spectral peak, 458 nm) promoted biofilm expansion, whereas green and red LED light (spectral peaks, 510 and 639 nm, respectively) did not (Fig. 3e). These observations demonstrate that the blue spectrum of low-level light (equivalent to the brightness of sunrise and sunset light or brightness under desk light) alters the growth and development of colony biofilms.
a Effect of light exposure on biofilm development. The WT strain was grown in MSgg under dark and moderate light (35 μmol m−2 s−1) conditions. Colony development was recorded every 24 h. The photographs on the far right show colony morphology at 120 h observed with a stereomicroscope. Scale bar, 5 mm. b The growth rate of colony biofilms. The areas of the three colonies are plotted for each condition. c Biofilm expansion in a half-shaded plate. The WT strain was grown in a plate, half of which was covered with aluminum foil, under moderate light conditions for 120 h. Scale bar, 5 mm. d Effect of light intensity on biofilm expansion. The WT strain was grown under low light (~14.5 mol m−2s−1) and dim light (~4.8 μmol m−2 s−1) for 120 h. e Effect of illumination with light of different spectra on biofilm expansion. The WT strain was grown under blue, green, and red LED light (~30 μmol m−2 s−1) for 120 h. These colony assays were performed three times with similar results.
We predicted that light exposure would promote biofilm expansion rather than flagellum-dependent colony expansion. In B. subtilis, biofilm expansion is driven by collective sliding motility, which depends on the lipopeptide surfactin and EPS components of biofilms, such as exopolysaccharides29,30,31. In support of this prediction, light-induced colony expansion was blocked by deletion of surfactin and exopolysaccharide biosynthesis genes (ΔsrfAC and ΔepsA operons, respectively) but not by deletion of a flagellin gene (Δhag) (Fig. 4a).
a Colony expansion is blocked by ΔsrfAC and ΔepsA-op mutations. The indicated strains were grown under dark and moderate light for 120 h. Cysteine and sodium L-ascorbate were added to a final concentration of 0.5 mM. The colony assays were performed three times with similar results. b Expression of PepsA-gfp in colony biofilms. The PepsA-gfp strain was grown in MSgg medium under dark and moderate light conditions, and expression of the PepsA-gfp reporter in the whole colony and at the colony edge was measured by flow cytometry. The WT strain without the reporter was used as a background control. Three independent colonies were analyzed for each assay.
The biofilm expansion phenotype suggested that light exposure may promote or prolong biofilm formation. To distinguish between these possibilities, we compared the expression of the epsA operon in whole colony biofilms between dark and moderate light conditions using the PepsA-gfp reporter. Note that culturing under moderate light had little or no effect on the GFP fluorescence of the constitutively expressing gfp reporter, Pspac-hy-gfp (Supplementary Fig. 4). Flow cytometry demonstrated that colony biofilms exhibited comparable bimodal expression of PepsA-gfp under dark and light conditions up to 72 h (Fig. 4b). However, at 96 h, the cell population highly expressing PepsA-gfp largely disappeared under dark conditions, whereas it was maintained from 72 h under light conditions. Since colony biofilms consist of slow- and fast-growing areas, PepsA-gfp expression was further examined in cells isolated from the fast-growing area, specifically the edge of colony biofilms. These cells also exhibited prolonged PepsA-gfp expression under light conditions (Fig. 4b). These results indicate that under dark conditions, biofilms grow and reach the mature phase, resulting in a reduction in expression of biofilm-forming genes. By contrast, under light conditions, biofilms continue to grow without entering the mature phase.
As biofilms mature, biofilm cells begin to sporulate16,32. We therefore determined whether sporulation is involved in light-induced biofilm expansion. To examine the effect of light exposure on sporulation, the sspB-gfp fusion gene was employed. Since the small acid-soluble spore protein SspB is induced in the late stage of sporulation and retained in mature spores33, the percentage of cells exhibiting fluorescence from the SspB-GFP fusion protein can be considered a proxy for sporulation frequency (Supplementary Fig. 5a). In the dark, over 80% of cells in colony biofilms exhibited SspB-GFP expression by 72 h (Supplementary Fig. 5b). In contrast to its strong impact on biofilm maturation, moderate light exposure only slightly reduced the cell population expressing SspB-GFP. Even under intense light exposure, 20% of cells still expressed SspB-GFP. Therefore, light exposure had only a weak inhibitory effect on sporulation. These results indicate that sporulation is unlikely to contribute to light-induced biofilm expansion.
In other non-photosynthetic bacteria, light exposure alters gene expression through the activation of photoreceptor proteins and/or the stimulation of ROS generation4,5,6,7,8,9,10,11,13,14. However, disruption of the photoreceptor gene ytvA or supplementation with the ROS scavengers, cysteine and ascorbic acid, did not prevent light-induced biofilm expansion (Fig. 4a). Furthermore, expression of ytvA and kinC is known to be subject to circadian control, with reduced gene expression occurring during light exposure21,22. However, expression of PytvA-gfp and PkinC-gfp reporters did not respond to light exposure under the present culture conditions (Supplementary Fig. 4). These observations indicate that light-induced biofilm expansion may be driven by an unknown mechanism that is independent of the YtvA photoreceptor and ROS generation.
Light inhibits the expression of pulcherriminic acid biosynthesis genes
In the dark, colony biofilms produced reddish-brown pigments (Fig. 3a). B. subtilis biofilms have been reported to secrete the iron chelator pulcherriminic acid, which binds to extracellular Fe3+ to form the reddish precipitates pulcherrimin, thereby limiting iron in the immediate environment34. Since biofilm formation requires large amounts of iron35, the production of pulcherriminic acid consequently leads to the arrest of biofilm growth34. The iron-limiting effect of pulcherriminic acid is believed to play a critical role in defending biofilm cells against ROS and oxidative stress in developed biofilms36,37. Pulcherriminic acid is synthesized by enzymes of the yvmC-cypX operon and subsequently exported by the specific transporter YvmA38,39,40 (Fig. 5a). Under the present culture conditions, ΔyvmC, ΔcypX, and ΔyvmA mutants all exhibited biofilm expansion independent of dark and light conditions (Fig. 5b). These observations suggest that light exposure may promote biofilm expansion by inhibiting pulcherriminic acid biosynthesis.
a Schematic representation of pulcherriminic acid biosynthesis. Proteins involved in the light response are highlighted in yellow. Speculative intracellular conversion of pulcherriminic acid to pulcherrimin and light-induced degradation of pulcherriminic acid are indicated in red and blue, respectively (see text). b Colony phenotype of gene mutations involved in pulcherriminic acid biosynthesis. Indicated strains were grown in MSgg for 120 h under dark and moderate light conditions. The ΔpchR mutant was grown in MSgg medium with 100 μM IPTG to induce yvmA expression. Scale bar, 5 mm. These colony assays were performed three times with similar results. c Light exposure suppresses pulcherrimin production. Pulcherrimin content per cell mass (A410/A600) was measured in 3-day-old colonies grown under dark (D) and light (L) conditions. Five different colonies were analyzed for each assay. Average and individual data are shown as bars and patches, respectively. Different letters indicate statistically significant differences, two-sided p values <0.05. (see Source Data). d Expression of the PyvmC-gfp reporter under different light conditions. The PyvmC-gfp strain was grown under dark and indicated light conditions. Expression of PyvmC-gfp in the whole colony was measured by flow cytometry. Three independent colonies were analyzed for each assay. Background controls are also shown for reference.
To test this possibility, the levels of biofilm-associated pulcherriminic acid and pulcherrimin were compared between dark and light conditions. Pulcherriminic acid and pulcherrimin were extracted collectively from 3-day-old colony biofilms as pulcherriminic acid using sodium hydroxide, and the levels of pulcherriminic acid were quantified using a spectrophotometer at 410 nm, which corresponds to the peak absorption wavelength of pulcherriminic acid41. Under dark conditions, the WT strain produced a measurable amount of pulcherriminic acid/pulcherrimin, whereas under light conditions, it produced little pulcherriminic acid/pulcherrimin, as did the pulcherriminic acid production-deficient ΔcypX mutant (Fig. 5c). These results indicate that light exposure exerts an inhibitory effect on pulcherriminic acid production.
The light response of non-photosynthetic bacteria is accompanied by transcriptional changes3. We therefore compared expression of the yvmC operon between dark and light conditions using the PyvmC-gfp reporter. Flow cytometry revealed that moderate light exposure resulted in a small and a large reduction in PyvmC-gfp expression at 24 and 48 h, respectively (Fig. 5d). This observation was corroborated by fluorescence microscopy (Supplementary Fig. 6). Fluorescence microscopy also demonstrated that PyvmC-gfp expression was more abundant at 48 h than at 24 h. To determine the extent of the light-induced reduction in PyvmC-gfp expression, the levels of the GFP protein levels in colony biofilms were quantified by SDS-PAGE. Quantitative analysis demonstrated that the reductions in the expression were 5.7-fold at 24 h and 21.8-fold at 48 h (Supplementary Fig. 7). Furthermore, since low-intensity light induced biofilm expansion (Fig. 3d), the effect of lower intensity light on PyvmC-gfp expression was examined. Low and dim light exposure also led to reduced PyvmC-gfp expression at 48 h (Fig. 5d). However, their impact was considerably less pronounced than that of moderate light.
Day/night cycles are a feature of the natural environment. To test whether expression of the yvmC operon responds to these cycles, WT and PyvmC-gfp reporter strains were cultured under cycles of 12 h dark/12 h light or 12 h light/12 h dark. Under both cycles, the WT strain formed expanded colony biofilms with brown stripes (Fig. 6a). In the advancing edge of these colony biofilms, PyvmC-gfp expression repeatedly rose and fell in response to alterations in dark and light periods under these cycles (Fig. 6b). However, the oscillation became less pronounced on day 4, perhaps due to nutrient depletion. These phenotypes indicate that pulcherriminic acid biosynthesis oscillates in dark/light or light/dark cycles, resulting in the formation of light brown stripes of pulcherrimin in expanded colony biofilms. Furthermore, the biofilm expansion phenotype showed that 12 h of darkness per day was insufficient to produce sufficient pulcherriminic acid to arrest colony expansion under the present culture conditions. A slightly longer period of darkness per day, i.e., 14 h dark/10 h light cycles, was required to prevent biofilm expansion under the present culture conditions (Fig. 6a). These results indicate that biofilm expansion involving pulcherriminic acid is regulated not only by light intensity but also by exposure time.
a Colony biofilms were grown under 12 h light/12 h dark, 12 h dark/12 h light, or 14 h dark/10 h light conditions. Image contrast was slightly increased to clearly visualize colony stripes. Scale bar, 5 mm. These colony assays were performed three times with similar results. b PyvmC-gfp expression under light/dark or dark/light cycles. The PyvmC-gfp strain was grown under 12 h light/12 h dark or 12 h dark/12 h light cycles. PyvmC-gfp expression at the edge of colony biofilms was measured at 9 h of each dark and light period, as indicated by the downward arrowheads in the diagrams. Three independent colonies were analyzed for each time point. c Response speed of PyvmC-gfp expression to changes in light conditions. PyvmC-gfp strain was grown under dark or light conditions for 24 h, and then the resulting colonies were transferred to opposite light conditions (time 0). After transfer, PyvmC-gfp expression was measured every hour by flow cytometry. PyvmC-gfp expression in colonies grown under constant dark or constant light conditions was used as references.
We determined the response speed of PyvmC-gfp expression to dark-to-light or light-to-dark transitions. To this end, the PyvmC-gfp reporter strain that had been cultivated under dark or light conditions for 24 h was transferred to opposite light conditions, and PyvmC-gfp expression was then monitored at hourly intervals. Changes in PyvmC-gfp expression were observed 2 h after the dark-to-light transition and 3 h after the light-to-dark transition (Fig. 6c). This demonstrates that the light response of PyvmC-gfp was considerably slower than that observed in photoreceptor-dependent systems in other bacteria42,43,44,45.
Light-responsive regulation of the yvmC operon
We investigated the mechanism of light-responsive expression of the yvmC operon. The yvmC operon is regulated by two repressors, the global regulator AbrB and the MarR family regulator PchR (Fig. 5a)46,47. The ΔabrB mutant exhibited increased PyvmC-gfp expression, yet its expression was reduced in response to light (Fig. 7). By contrast, the ΔpchR mutant, in which yvmA, the downstream gene of pchR, was constitutively expressed from the spac promoter28, exhibited increased PyvmC-gfp expression independent of dark and light conditions. These observations indicate that PchR is responsible for the light-responsive expression of the yvmC operon. However, despite the constitutive expression of the yvmC operon and yvmA, the ΔpchR mutant exhibited biofilm expansion with no pulcherrimin pigment under light (Fig. 5b). In fact, the ΔpchR mutant produced little pulcherriminic acid/pulcherrimin under light (Fig. 5c). These results indicate that the reduction in pulcherrimin biosynthesis in response to light occurs via a post-transcriptional mechanism rather than via light-responsive expression of the yvmC operon.
WT and PyvmC-gfp reporter strains were grown under dark and moderate light conditions. Expression of PyvmC-gfp was measured by flow cytometry. Peak positions of PyvmC-gfp expression in the WT strain under dark and light conditions are indicated by blue and orange vertical lines, respectively. Strains carrying the ΔpchR mutation were grown in MSgg medium with 100 μM IPTG to induce yvmA expression. For each assay, three independent colonies were analyzed.
Since the light-responsive expression of the yvmC operon was not the primary cause of the reduction in pulcherriminic acid/pulcherrimin levels, we hypothesized that upon light exposure, pulcherriminic acid levels would initially decline, leading to the repression of yvmC operon expression by PchR. To test this hypothesis, we examined the effect of pulcherriminic acid levels on PyvmC-gfp expression (Fig. 7). ΔyvmC and ΔcypX mutants, which are deficient in pulcherriminic acid biosynthesis, exhibited a significant reduction in PyvmC-gfp expression under both dark and light conditions. By contrast, the ΔyvmA mutant, which accumulates pulcherriminic acid intracellularly due to a defect in its export48, exhibited light-responsive expression of PyvmC-gfp comparable to that of the WT strain, but with higher expression levels, particularly at 48 h. Thus, a loss of pulcherriminic acid biosynthesis and an increase in intracellular pulcherriminic acid decreased and increased PyvmC-gfp expression, respectively. The phenotypes of ΔcypX and ΔyvmA mutants were suppressed by the ΔpchR mutation, as ΔcypX ΔpchR and ΔyvmA ΔpchR double mutants overexpressed PyvmC-gfp to a similar extent as the ΔphcR mutant. These results indicate that pulcherriminic acid may be involved in the regulation of yvmC operon expression via PchR, which provides support for the hypothesis. Furthermore, a tenfold reduction in iron supplementation resulted in a delay in PyvmC-gfp expression, which was suppressed by the ΔpchR mutation (Fig. 7, 1/10 FeCl3). This suggests that pulcherrimin (Fe3+-bound pulcherriminic acid) rather than pulcherriminic acid may be required for expression of the yvmC operon.
To test whether pulcherriminic acid or pulcherrimin functions as an effector of PchR, we performed electrophoretic mobility shift assays using purified C-terminal His-tagged PchR, FITC-labeled yvmC promoter DNA containing a PchR-binding site, pulcherriminic acid, and iron. Pulcherriminic acid was purified from WT biofilms. As previously described46, PchR bound to yvmC promoter DNA and formed the PchR-DNA complex (Fig. 8a). Pulcherriminic acid and FeCl3, when used individually, did not affect complex formation. However, when used in combination, they inhibited it. In the presence of pulcherriminic acid, FeCl2 also inhibited complex formation, but its effect was much weaker than that of FeCl3 (Fig. 8b), which is consistent with pulcherriminic acid being a chelator of Fe3+. These results indicate that pulcherrimin acts as an effector of PchR, thereby impeding the binding of PchR to the yvmC promoter.
a Electrophoretic mobility shift assay of PchR binding to the yvmC promoter. The PchR-6×His protein was incubated with FITC-labeled yvmC promoter DNA in the presence (+) or absence (−) of indicated concentrations of pulcherriminic acid and FeCl3. Free DNA and the PchR-DNA complex are indicated by arrows. b Fe3+ has a stronger inhibitory effect on DNA binding of PchR in the presence of pulcherriminic acid than Fe2+. These experiments were performed at least twice with similar results.
However, in a reducing cytoplasmic environment, the predominant form of iron is Fe2+, and any Fe3+ present is short-lived and rapidly reduced to Fe2+. This raised the question of whether Fe3+ is available for pulcherrimin formation in the cytoplasm of biofilm cells. To address this question, the ΔpchR-yvmA mutant was employed, which overexpresses the yvmC operon but is unable to export pulcherriminic acid due to the absence of the YvmA exporter. If Fe3+-generating processes are active in the cytoplasm, the accumulated pulcherriminic acid within the cytoplasm would chelate these Fe3+ ions, forming pulcherrimin. A high concentration of pulcherrimin would then undergo self-assembly into insoluble supramolecules, namely pulcherrimin precipitates49. To ascertain the formation of pulcherrimin precipitates within ΔpchR-yvmA mutant cells, cell lysates prepared from colony biofilms were separated into supernatant and precipitate fractions by centrifugation. Subsequently, the levels of pulcherriminic acid/pulcherrimin in each fraction were quantified. In this experiment, the ΔcypX mutant was used as a background control. The results demonstrated that the 24-h-old colony biofilms of the ΔpchR-yvmA mutant produced a discernible amount of supernatant pulcherriminic acid/pulcherrimin, yet only a small amount of pulcherrimin precipitates (Supplementary Fig. 8a). By contrast, the 48-h-old colony biofilms did not produce a discernible amount of supernatant pulcherriminic acid/pulcherrimin but did exhibit a notable accumulation of pulcherrimin precipitates. The accumulation of pulcherrimin precipitates was predicted to disrupt the Fe2+/Fe3+ cycle, resulting in a reduction in both Fe2+ and Fe3+ levels within the cytoplasm. Subsequently, the reduction in Fe2+ levels would induce expression of iron limitation-responsive genes, such as the dhbA operon50. Consistent with the prediction, the accumulation of pulcherrimin precipitates was accompanied by induction of the PdhbA-gfp reporter (Supplementary Fig. 8b). This induction was indeed dependent on the intracellular accumulation of pulcherrimin, as evidenced by the lack of the induction in the WT and ΔpchR strains. These results indicate that Fe3+-generating processes are active in the cytoplasm, particularly in that of developed biofilms, allowing for the formation of pulcherrimin in the cytoplasm. This is likely consistent with the observation that yvmC operon expression was more abundant at 48 h than at 24 h (Supplementary Fig. 6). The lack of induction of the PdhbA-gfp reporter in the WT and ΔpchR strains also indicates that the majority of pulcherriminic acid is exported from the cytoplasm immediately after synthesis in the presence of the YvmA exporter. Thus, only a minor fraction of pre-secreted pulcherriminic acid may form pulcherrimin for yvmC operon expression in the WT strain.
Pulcherriminic acid is sensitive to light
Next, we attempted to identify the primary cause of the reduction in pulcherriminic acid levels under light conditions. Pulcherriminic acid is synthesized from leucyl tRNA, and leucine is synthesized from pyruvate via Ile and Leu biosynthetic pathways (Fig. 5a). To test whether light exposure inhibits these biosynthetic pathways, resulting in the reduction in pulcherriminic acid production and yvmC operon expression, we employed the ΔleuD mutant, which produces pulcherriminic acid from extraneously supplied leucine, independently of the activities of Ile and Leu biosynthesis. The ΔleuC mutant exhibited light-responsive expression of PyvmC-gfp (Fig. 7), thus refuting this possibility.
We next tested whether light exposure affects the stability of YvmC and CypX. To quantify the levels of YvmC and CypX proteins, C-terminal 3×FLAG-tagged YvmC and C-terminal T7-tagged CypX were expressed from the spac-hy promoter51 in ΔyvmC and ΔcypX mutants, respectively. Peptide tagging and constitutive expression of YvmC and CypX did not affect these protein functions, as yvmC-3×flag and cypX-t7 strains exhibited light-induced biofilm expansion comparable to that of the WT strain (Supplementary Fig. 9a). Western blotting revealed that light exposure did not reduce YvmC-3×FLAG and CypX-T7 protein levels (Supplementary Fig. 9b). Therefore, light exposure is unlikely to destabilize YvmC or CypX.
The cytochrome P450 family protein CypX requires electrons to synthesize pulcherriminic acid40, and these electrons are provided by the three-component electron transfer system, which consists of ferredoxin, flavodoxin, and ferredoxin/flavodoxin NADP+ reductase52. This electron transfer system also provides electrons to other reducing enzymes, including the membrane phospholipid desaturase Des, which forms unsaturated fatty acids53. Because the Δdes mutation increases des transcription54, impairment of the three-component electron transfer system was expected to increase des transcription. However, the Pdes-gfp reporter strain exhibited comparable expression under both dark and light conditions (Supplementary Fig. 10), suggesting that light exposure is unlikely to affect the electron transfer system.
The data so far provide no evidence that light exposure inhibits pulcherriminic acid biosynthesis. We next investigated whether light exposure results in the degradation of pulcherrimin or pulcherriminic acid. To this end, purified pulcherrimin and pulcherriminic acid were exposed to moderate light. Light exposure did not affect pulcherrimin because pulcherrimin was converted to similar amounts of pulcherriminic acid in sodium hydroxide with or without 60 min of light exposure (Fig. 9a). To assess the photostability of pulcherrimin under culture conditions, the WT strain was inoculated in proximity to a WT colony that had been grown in the dark for three days to form pulcherrimin. The plate was then incubated under moderate light (Fig. 9b). The newly formed WT colony exhibited biofilm expansion, whereas the WT colony that had already produced pulcherrimin did not. Furthermore, the expansion of the newly formed WT colony was inhibited in the vicinity of the pulcherrimin-produced WT colony. These results indicate that pulcherrimin is photostable and does not release iron under light culture conditions.
a Light exposure does not affect pulcherrimin. A pulcherrimin suspension was incubated for 60 min in the dark or moderate light. The suspension was then mixed with 2 M NaOH to convert pulcherrimin to pulcherriminic acid, and the pulcherriminic acid content was measured with a spectrophotometer at 410 nm. Three samples were analyzed for each assay. Average and individual data are shown as bars and patches, respectively. There is no statistically significant difference (two-sided p value, 0.73). b Pulcherrimin is photostable under culture conditions. The WT strain was inoculated near a WT colony that had been cultivated in the dark for 72 h to produce pulcherrimin. Thereafter, the plate was incubated under moderate light for 120 h. New and existing colonies are indicated by “new” and “old”, respectively. c Light sensitivity of pulcherriminic acid. Pulcherriminic acid solution in a 96-well plate with a cover was incubated under moderate light. Before and after 10, 30, and 60 min of light exposure, absorption spectra from 210 to 500 nm were recorded with a spectrophotometer. Two sets of data were presented for each time point, although the two plots for each time point are nearly superimposable. d LC-MS chromatogram (150–500 m/z, positive ion detection) of pulcherriminic acid solution before and after 10 and 60 min of light exposure. e The increase in iron supplementation does not affect PyvmC-gfp expression. The PyvmC-gfp reporter strain was grown in MSgg media with indicated concentrations of FeCl3. Expression of PyvmC-gfp under dark or light conditions was measured by flow cytometry. f The increase in iron supplementation diminishes the impact of light on pulcherrimin levels. Pulcherrimin content per cell mass (A410/A600) was measured in 3-day-old colonies grown in MSgg media supplemented with indicated concentrations of FeCl3. Five different colonies grown under dark (D) or light (L) conditions were analyzed for each assay. Average and individual data are shown as bars and patches, respectively. Two-sided p values between dark and light conditions are less than 0.05 for all three FeCl3 concentrations (see Source Data).
Before exposure to moderate light, purified pulcherriminic acid exhibited three distinct absorption spectra at 240, 280, and 410 nm, as previously described41, although the 240 nm peak was partially affected by impurities (Fig. 9c). These absorption peaks became smaller after 10 min of light exposure and almost disappeared after 60 min of light exposure. By contrast, incubation for 60 min in the dark had no effect on pulcherriminic acid (Supplementary Fig. 11). To further explore the impact of light exposure on pulcherriminic acid, a liquid chromatography-photodiode array-mass spectrometry (LC/PDA-MS) analysis was conducted using commercially available pulcherriminic acid. The analysis revealed that after 10 min and 60 min of light exposure, a peak of pulcherriminic acid (m/z 257.2) almost disappeared (Fig. 9d), and two novel peaks of smaller products (m/z198.2 and m/z 229.1) emerged instead (Supplementary Fig. 12). These results indicate that light exposure triggers the photolysis of pulcherriminic acid, which may result in the cleavage or structural alteration of its six-membered ring—the structural element responsible for light absorption (see Fig. 5a for the structure of pulcherriminic acid).
To test whether photolysis of pulcherriminic acid occurs in colony biofilms, we took advantage of the difference in photostability between pulcherriminic acid and pulcherrimin. Since an increase in iron supplementation in the medium facilitates pulcherrimin formation36, we hypothesized that if the photolysis of pulcherriminic acid occurs under light culture conditions, an increase in iron supplementation would suppress its photolysis by accelerating the conversion of photosensitive pulcherriminic acid to photostable pulcherrimin. However, as intracellular iron levels are strictly regulated by both iron uptake and efflux systems55, suppression of photolysis would only occur outside cells. To test this hypothesis, PyvmC-gfp expression and pulcherriminic acid/pulcherrimin levels were measured in WT colony biofilms grown in MSgg medium (containing 50 μM FeCl3) and in iron-enriched MSgg media containing 150 μM or 500 μM FeCl3. Expression of PyvmC-gfp was comparable in the three media under dark conditions (Fig. 9e). Under moderate light, PyvmC-gfp expression was reduced in all three media to the same extent. As PyvmC-gfp was responsive to intracellular pulcherrimin, these observations indicate that an increase in FeCl3 does not affect the intracellular levels of pulcherrimin, which rules out the possibility that increased FeCl3 affects pulcherriminic acid biosynthesis. By contrast, biofilm-associated pulcherriminic acid/pulcherrimin gradually increased with an increase in FeCl3 in the medium under dark conditions (Fig. 9f). Moderate light exposure resulted in a reduction in pulcherriminic acid/pulcherrimin levels in all three media. However, the impact of light exposure diminished with the increase in FeCl3. Specifically, the light/dark ratios of pulcherriminic acid/pulcherrimin levels were 18.3, 25.9, and 41.6% in 50 μM FeCl3, 150 μM FeCl3, and 500 μM FeCl3, respectively. These observations support the hypothesis that an increase in iron supplementation suppresses the photolysis of pulcherriminic acid in the extracellular environment. While in vivo evidence for the intracellular photolysis of pulcherriminic acid is still lacking, the observation that light exposure did not affect pulcherriminic acid biosynthesis indicates that the photolysis of pulcherriminic acid is the most probable mechanism for the light response within cells. Taken together, these results indicate that the photosensitive property of pulcherriminic acid enables it to control iron availability, transcription, and biofilm development in response to changes in light conditions.
Discussion
The present study demonstrates that B. subtilis biofilms have a distinctive light-response mechanism that is mediated by the iron chelator pulcherriminic acid. In the absence of light, the induction of pulcherriminic acid production resulted in limiting iron levels, which directed the biofilms toward the mature phase. In the presence of light, the levels of pulcherriminic acid decreased, leading to the continuous growth of the biofilms. Thus, pulcherriminic acid modulates biofilm development in response to dark and light. The reduction in pulcherriminic acid levels was also accompanied by a reduction in yvmC operon expression. EMSA analysis indicated that intracellular pulcherrimin probably functions as an effector of the PchR repressor to induce yvmC operon expression in the absence of light. However, the light response of pulcherriminic acid was regulated at the post-transcriptional level. Purified pulcherriminic acid underwent photolysis in vitro, a property of which was verified under culture conditions. Based on these findings, we propose that the photosensitive property of pulcherriminic acid enables it to control iron availability, transcription, and biofilm growth in response to light. Furthermore, the finding that intense light exposure inhibited colony formation by exciting protoporphyrin IX under iron-limited conditions indicates that the reduction in pulcherriminic acid levels in response to light may also serve as a mechanism to prevent iron limitation under light conditions.
Pulcherriminic acid is known to play a critical role in defense against ROS and oxidative stress in B. subtilis biofilms36,37. During biofilm development, levels of ROS and oxidative stress are elevated, resulting in induction of the DNA damage response and increases in mutation rates, cell death, and eDNA release56,57,58,59. In this situation, the iron-limiting effect of pulcherriminic acid is thought to suppress ROS generation by inhibiting the Fenton/Haber–Weiss reaction, thereby protecting biofilm cells from ROS and oxidative stress36,37. Indeed, a pulcherriminic acid-deficient mutant exhibits altered expression of hundreds of genes, including induction of the DNA damage response in biofilms36. Therefore, the finding that the biofilms produced little pulcherriminic acid under light conditions was surprising and indicated that this light response likely serves a biological purpose. The light response of pulcherriminic acid was induced by low-level light, which is equivalent to the intensity of light at sunrise and sunset. However, unlike intense light, low light did not have a deleterious effect on B. subtilis (Fig. 1), indicating that there is no urgent need to respond to low-level light. In the natural environment, the light response of pulcherriminic acid would be activated in biofilms growing in bright environments, such as soil surfaces. However, given the limited capacity of B. subtilis to survive in direct sunlight, these biofilms may thrive in bright, but not direct sunlight, environments. Even in these environments, light intensity varies with the time of day. Consequently, biofilms exposed to weak light in the morning would be exposed to brighter light in the afternoon. We demonstrated that intense light, which is approximately equivalent to the brightness on a cloudy day, induced ROS generation by exciting protoporphyrin IX under iron-limited conditions (Fig. 2c, f). It is therefore reasonable to predict that the production of the iron chelator pulcherriminic acid in daylight conditions probably induces deleterious levels of ROS through this mechanism, which may pose a greater threat to biofilm cells than ROS generation by the Fenton/Haber–Weiss reaction under daylight conditions. If this is indeed the case, the reduction in pulcherriminic acid in response to weak light may serve as a mechanism to prevent iron limitation during bright daytime. Some non-photosynthetic bacteria have been reported to utilize weak morning light as an anticipatory cue for the induction of gene expression that enables them to adapt to subsequent daytime dry conditions10,14. Likewise, B. subtilis biofilms may employ a light-response strategy analogous to these bacteria, and thus, the reduction in pulcherriminic acid in response to weak light may be an anticipatory response for adaptation to subsequent brighter daytime conditions. Further research is required to determine the precise function of the light response of pulcherriminic acid. Light-dependent control of iron metabolism has also been reported in human pathogens, including Actinetobacter baumannii60,61. In the light, the iron limitation response in these bacteria is inhibited even under iron-limited conditions. This is the complete opposite of the light response of B. subtilis biofilms, which prevents iron limitation in light. These different responses of iron metabolism to light in these bacteria may be due to differences in their habitat or lifestyle.
The light-induced reduction in pulcherriminic acid levels delays the maturation of biofilms, thereby facilitating the continuous expansion of biofilms. Biofilm expansion is the most powerful surface movement mechanism in B. subtilis under low water conditions31,32,33. In 1.5% agar medium, in which flagellar motility is unable to facilitate movement62, the expansion rate of biofilms reaches several millimeters per day. Although biofilm movement in agar medium differs from that in soil environments, a few millimeters of vertical movement is probably sufficient for the expanding edges of biofilms to reach dark areas in soil. Since intense light exposure reduced sporulation, the movement to dark areas may increase the probability of survival. Future studies will be needed to examine the effect of pulcherriminic acid on biofilm growth in natural environments.
The expression of the yvmC operon was reduced in response to light. This response was dependent on pulcherriminic acid biosynthesis, as evidenced by the inhibitory effect of ΔyvmC and ΔcypX mutations on yvmC operon expression (Fig. 6). EMSA analysis revealed that pulcherriminic acid inhibited the binding of PchR to yvmC promoter DNA in the presence of Fe3+. These findings indicate that some of the pre-secreted pulcherriminic acid binds to Fe3+ to form pulcherrimin, which functions as an effector of PchR to induce yvmC operon expression. This regulatory mechanism establishes a positive feedback loop that regulates yvmC operon expression (Fig. 5a). It seems reasonable that B. subtilis uses pulcherrimin as an effector, rather than pulcherriminic acid, because the involvement of this iron-containing compound in yvmC operon expression would not only prevent the production of pulcherriminic acid under iron-limited conditions but also limit the production of pulcherriminic acid to a level that does not cause iron starvation. However, the question remains as to whether the reducing environment of the cytoplasm provides sufficient Fe3+ for pulcherrimin formation. We demonstrated that the cytoplasmic accumulation of pulcherriminic acid resulted in the formation of pulcherrimin precipitates within 48-h-old cells but not within 24-h-old cells (Supplementary Fig. 8a), indicating that a portion of the free iron within cells of developed biofilms exists in the Fe3+ state. Given that cells encounter elevated levels of ROS in developed biofilms36,37, ROS generation may serve as a source for the generation of short-lived Fe3+ in the cytoplasm. If this is the case, expression of the yvmC operon would be increased with increasing ROS levels, enhancing the protective function of pulcherriminic acid against ROS36,37. Pulcherrimin undergoes multimerization after binding to ferric iron, resulting in supramolecular precipitates in the extracellular environment49. Cytoplasmic pulcherrimin might also be expected to form precipitates within cells; however, the overproduction of pulcherriminic acid by the ΔpchR mutation did not result in iron limitation, suggesting that the majority of pulcherriminic acid is exported immediately after synthesis, which would maintain the amount of cytoplasmic pulcherrimin at a low level. It has been reported that intracellular concentrations of typical repressors and leucine (the source of pulcherriminic acid) are in the range of a few hundred molecules per genome and <300 μM, respectively63,64. Therefore, the concentrations of pulcherrimin involved in transcriptional regulation may be very low and not sufficient for the formation of supramolecular pulcherrimin within cells.
Despite the constitutive expression of the yvmC operon, the ΔpchR mutant produced little pulcherrimin in light (Fig. 5c), suggesting that the light response of pulcherriminic acid production is primarily regulated at the post-transcriptional level. However, we obtained no evidence to show that light exposure destabilizes YvmC and CypX or interferes with the electron transfer system for CypX. Instead, we observed the gradual disappearance of pulcherriminic acid under light exposure in vitro, accompanied by the loss of its light-absorbing property. (Fig. 9c, d). Given that conjugated double bonds are responsible for light absorption in spectrophotometry, these findings indicate that pulcherriminic acid is subject to photolysis, resulting in the cleavage or alteration of its six-membered ring. The light response of B. subtilis biofilms was elicited by blue LED light (spectral peak, 458 nm). Moreover, pulcherriminic acid exhibits blue light absorption, including at a wavelength of 410 nm. Based on these observations, we predict that blue spectrum light may excite electrons in pulcherriminic acid, which may consequently cause its photolysis.
Photolysis is not only a response regulating the light response of pulcherriminic acid, but also plays a role in regulating the light response of cobalamin (vitamin B12)-dependent photoreceptors65. CarH-type photoreceptors use adenosylcobalamin (AdoCbl) as the light-sensing chromophore to mediate light-dependent gene regulation. In the dark, AdoCbl-bound CarH forms a tetramer that binds to the promoter region of the carotenoid biosynthesis operon to repress its transcription in Myxococcus xanthus65. Upon exposure to light, the photosensitive Co-C bond of AdoCbl is cleaved, releasing 4’,5’-anhydroadenosine from AdoCbl. The resulting bis-His cobalamin (bis-HisCbl)-bound CarH undergoes structural reorientation that results in tetramer disassembly, DNA dissociation, and subsequent transcription activation of the carotenoid biosynthesis operon. In this manner, the photosensitive molecule AdoCbl exerts direct effects on the DNA-binding activity of CarH. However, unlike AdoCbl, pulcherriminic acid exerts its regulatory effect on the DNA-binding activity of the PchR repressor indirectly, through the formation of pulcherrimin. Consequently, the light response of the yvmC operon expression requires a reduction in the cellular pulcherrimin level through the photolysis of pulcherriminic acid. This may be the reason why the light response of the yvmC operon expression was considerably slower than that of other photoreceptor-dependent systems (Fig. 6c).
Based on the findings, we propose a model for the light response mechanism of B. subtilis biofilms as follows (Fig. 5a). Upon exposure to light, pulcherriminic acid undergoes photolysis, resulting in a reduction in pulcherriminic acid levels and yvmC operon expression. The reduction in pulcherriminic acid levels not only disables the growth arrest mechanism of biofilms but also prevents the production of deleterious ROS via protoporphyrin IX. The continuous growth of biofilms allows the expanding edge of biofilms to reach dark areas, which increases the probability of survival. Pulcherrimin production has been reported in the yeast Metschnikowia pulcherrima66,67. Furthermore, the genes responsible for pulcherriminic acid synthesis are also conserved in isolates of the Bacillales, including the opportunistic pathogen Bacillus cereus and the skin commensal bacterium Staphylococcus epidermidis33. It will be of interest to determine whether pulcherriminic acid plays a role in the light response of these and other bacteria.
Methods
Bacterial strains and media
The B. subtilis strain NCIB 3610 and its derivatives used in this study are listed in Supplementary Table S1. Since strain NCIB 3610 has low competence ability, mutant alleles were first introduced into the domesticated strain 168 and then transferred to strain NCIB 3610 by transformation with chromosomal DNA. Since strain 168 has several mutations that affect biofilm formation, the possibility of introducing these unwanted biofilm defects into strain NCIB 3610 was eliminated by examining the colony morphology of the transformants. In the construction of double and triple mutants, the mutant alleles were transferred to other NCIB 3610-derived strains via transformation using chromosomal DNA. Primers used for strain construction are listed in Supplementary Table S2. B. subtilis strains were grown in LB [LB Lennox; 10 g/L bacto-tryptone (Thermo Fisher, MA, USA), 5 g/L bacto-yeast extract (Thermo Fisher), 5 g/L NaCl], MSgg [5 mM potassium phosphate (pH 7), 100 mM MOPS (pH 7), 2 mM MgCl2, 700 μM CaCl2, 50 μM MnCl2, 50 μM FeCl3, 1 μM ZnCl2, 2 μM thiamine, 0.5% (w/v) glycerol, 0.5% (w/v) glutamate, 50 μg/mL tryptophan]16, or DSM [8 g/L bacto-nutrient broth (Thermo Fisher), 1 g/L KCl, 0.12 g/L MgSO4·7H2O, 1 mM Ca(NO3)2, 10 μM MnCl2, 1 μM FeSO4]. E. coli strains JM105 and JM109 were used for plasmid construction and maintenance.
Growth conditions
B. subtilis strains grown overnight at 30 °C in LB were serially diluted in LB for light sensitivity assays and 100-fold diluted for expression analysis and biofilm assays. Two microliters of the dilutions were spotted on LB or MSgg solid medium and grown under dark or light conditions at 30 °C. Intense and moderate light (110 and 35 μmol m−2s−1, respectively) was used in the light sensitivity assays, while moderate light was used in biofilm assays. Under intense and moderate light conditions, culture plates were placed under 18 W fluorescent lamps FL20SSECW18XF2 (Panasonic, Osaka, Japan). Under low and dim light conditions (14.5 and 4.8 μmol m−2s−1, respectively), culture plates were placed under 8 W fluorescent lamps FL8D (NEC, Tokyo, Japan). For spectrum selectivity testing, 7 W LED bulbs [blue, LDA7B-C50; green, LDA7RG-C50; red, LDA7R-C50 (Beamtec, Saitama, Japan), 20 μmol m−2s−1] were used. Brightness (μmol m−2s−1) was measured using a Sekonic C-7000 spectrometer (Sekonic, Tokyo, Japan).
Transposon mutagenesis
The temperature-sensitive plasmid pMarA carrying Kmr TnYLB68 was introduced into the WT strain. Twelve independent colonies were grown for 18 h at 28 °C in 5 mL LB supplemented with 10 μg/mL kanamycin. About 0.5 mL of the cultures were added to 4.5 mL LB and further cultured at 43 °C for 2 h. This process was repeated once more. After centrifugation at 5800 × g for 5 min, the cells were resuspended in the same volume of LB 20% glycerol and stored at −80 °C. One hundred microliters of the 100-fold diluted stocks were spread on a plate of LB solid medium supplemented with kanamycin, and 71 light-resistant colonies were isolated from approximately 20,000 Tn insertions under intense light at 30 °C. To identify true light-resistant transposon insertions, backcross transformation was performed using chromosomal DNA isolated from light-resistant colonies. For eleven Tn insertions with 100% linkage between Kmr and light resistance, their insertion sites were determined according to the procedure described previously68.
Expression of gfp reporters
Colonies were grown in LB or MSgg medium as described above. An entire colony was scraped with an inoculation loop and suspended in 600 μL PBS (81 mM Na2HPO4, 26.8 mM KCl, 14.7 mM KH2PO4, and 1.37 mM NaCl). For analysis of the advancing edge colony, ~2 to 3 mm of the edge region of the colony was scraped and suspended in 600 μL PBS. After dispersing cell aggregates by gentle sonication, the suspensions were used for flow cytometry analysis. If necessary, cells were fixed with 4% paraformaldehyde and stored at 4 °C as previously described32. Single-cell fluorescence was measured using a BD Accuri C6 flow cytometer (BD Biosciences, New Jersey, USA). Fifty thousand events were recorded per sample. The threshold for FSC-H was set at 20,000 to remove signals from cell debris and contaminants. Three independent colonies were analyzed per assay. Histograms were generated using BD Accuri C6 software (BD Biosciences).
Fluorescent microscopy
Cells were observed using a BX53 microscope (Olympus, Tokyo, Japan) equipped with an AdvanCam-E3Rs digital color camera (Advan Vision, Tokyo, Japan). To quantify the intensity of GFP fluorescence in individual cells, the images were analyzed using the MicrobeJ plugin of the ImageJ software69.
GFP fluorescence in SDS-PAGE
Colonies grown on LB or MSgg were suspended in 700 μL PBS supplemented with 10 mM EDTA (pH 8.0) and 2 mM phenylmethylsulfonyl fluoride (PMSF). After mild sonication to disperse cell aggregates, samples were diluted to an OD600 of 3.0. Diluted samples (600 μL) were mixed with 7 μL 10% SDS and sonicated to lyse the cells. After centrifugation at 15,100 × g for 5 min, 10 μL of supernatant was separated by SDS-PAGE (SuperSep Ace 10–20%, FUJIFILM Wako Pure Chemical, Osaka, Japan) without prior heating of the samples. After electrophoresis, GFP fluorescence images were captured with FUSION FX (Vilber, Marne-la-Vallée, France). The gels were then stained with Coomassie Brilliant Blue R250, and the stained images were captured using FUSION FX. The gel images were quantitatively analyzed using Evolution Capt (Vilber).
Measurement of pulcherriminic acid and pulcherrimin contents
To quantify biofilm-associated pulcherriminic acid and pulcherrimin, a 3-day-old colony was dissolved in 600 μL PBS, and the cells were dispersed by gentle sonication. OD600 of the cell suspension was measured using a spectrophotometer (U-1800; Hitachi High-Tech Corporation, Tokyo, Japan). The cell suspension was well mixed with the same volume of 2 M NaOH, and pulcherrimin was extracted as pulcherriminic acid. After centrifugation at 15,100 × g for 3 min, the supernatant was used for OD410 measurement. Pulcherriminic acid/pulcherrimin contents were determined as the average of OD410/OD600 ratios of five independent samples.
To quantify the intracellular pulcherriminic acid and pulcherrimin of the ΔpchR-yvmA mutant, the cell suspensions prepared from 1-day-old and 2-day-old colonies were treated with 0.5 mg/ml lysozyme at 37 °C for 10 min, and then the cells were lysed by sonication. The resulting cell lysates were separated into supernatant and precipitate fractions by centrifugation at 15,100 × g for 5 min. The supernatant fractions were mixed with an equal volume of 2 M NaOH, while the precipitate fractions were dissolved in twice the volume of 1 M NaOH. After centrifugation at 15,100 × g for 3 min, the supernatants were used for OD410 measurement.
Purification of pulcherriminic acid
The WT strain was grown statically in 200 mL MSgg supplemented with FeCl3 (final 150 μM) at 37 °C for 4 days. The culture was centrifuged at 5800 × g for 5 min. Purification of pulcherrimin was performed as previously described70. The precipitates containing cells and pulcherrimin were dissolved in 5 mL of 2 M NaOH, and the cells and hydrated iron oxide formed were removed by centrifugation at 5800 × g for 5 min. The supernatant containing pulcherriminic acid was adjusted to pH 1.0 with HCl, mixed with FeCl3 (final 2 mM), and stored at 4 °C overnight. The pulcherrimin precipitate was then collected by centrifugation at 5800 × g for 5 min and washed three times with 5 mL deionized water. The water-suspended pulcherrimin was aliquoted and precipitated, and the pulcherrimin precipitate was stored at −30 °C. The pulcherrimin precipitate was dissolved in an appropriate volume of 0.2 M NaOH to separate it into pulcherriminic acid and iron, and the resulting hydrated iron oxide was removed by centrifugation at 15,100 × g for 5 min. The pH of the supernatant was adjusted to 7.6 with 1 M MOPS and used as pulcherriminic acid in experiments.
Expression and purification of PchR-6×His
The pchR coding region was amplified by PCR using primers pchR-F1-3 and pchR-R1-2 (Supplementary Table S2). The 5’ ends of these primers contain 20 nt sequences overlapping with those of pET22b. The PCR products and NdeI/XhoI-digested pET22b were ligated using Gibson assembly master mix (New England Biolabs, Massachusetts, USA). The resulting plasmid pETpchR was introduced into E. coli BL21(DE3) pLysS. One transformant was cultured in 200 mL of autoinduction medium [12 g/L bacto-tryptone (Thermo Fisher), 24 g/L bacto-yeast extract (Thermo Fisher), 2.2 g/L KH2PO4, 9.4 g/L K2HPO4, 0.6% (w/v) glycerol, 0.5% (w/v) glucose, 0.2% (w/v) lactose] supplemented with 50 μg/mL ampicillin at 25 °C for 24 h. Cells were harvested by centrifugation at 5800 × g for 5 min and suspended in binding buffer [20 mM sodium phosphate (pH 7), 500 mM NaCl, 5 mM imidazole, 1 mM PMSF]. The cells were lysed by sonication, and the cell lysate was clarified by centrifugation three times at 5800 × g for 5 min. The supernatant was mixed with 1 mL of Talon metal affinity resin (Takara bio, Shiga, Japan) in a roller for 30 min at 4 °C. The protein-resin mixture was applied to a Poly-Prep chromatography column (Bio-Rad, CA, USA) and washed with 100 mL of binding buffer. PchR-6×His was eluted with a stepwise imidazole gradient (20 to 160 mM). The eluate fractions containing PchR-6×His were combined and dialyzed sequentially against 20 mM Tris-HCl (pH 7.6), 100 mM KCl, 1 mM MgCl2 and 20 mM Tris-HCl (pH 7.6), 100 mM KCl, 1 mM MgCl2, 50% glycerol. The PchR-6×His solution was aliquoted and stored at −80 °C. Protein concentration was determined using Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad).
EMSA
The yvmC promoter region containing a PchR-binding site was amplified by PCR using FITC-labeled pchR-F10 and pchR-R10 primers (Supplementary Table S2). PCR products were purified using QIAquick PCR Purification Kit (Qiagen, Venlo, the Netherlands). The DNA-binding assay mixture contained the following components in 12 μL: 20 mM Tris-HCl (pH 7.6), 100 mM KCl, 1 mM MgCl2, 50 μg/mL BSA, 50 μg/mL poly(dI-dC), 1.3 μL PCR DNA, and indicated concentrations of PchR-6×His. Pulcherriminic acid solution was prepared using 0.2 M NaOH and 1 M MOPS as described above. To reduce the ionic strength, the solution was diluted tenfold or more with water, and 2 μL of the dilutions were added to the assay solutions. After incubation at room temperature for 15 min, the reaction mixtures were subjected to 5% nondenaturing polyacrylamide gel electrophoresis. DNA bands were visualized using a FUSION FX chemiluminescence imaging system (Vilber, Marne-la-Vallée, France). Note that pulcherriminic acid concentrations were determined based on the dry weight of pulcherrimin, and the calculated concentration may be overestimated due to impurities.
Western blot analysis of YvmC-T7 and CypX-T7 proteins
Four colonies grown for 24 h on MSgg medium were suspended in 700 μL PBS supplemented with 5 mM EDTA (pH 8.0) and 1 mM PMSF. After dispersing cell aggregates by gentle sonication, the samples were diluted to an OD600 of 3. About 600 μL of the diluted samples were mixed with 7 μL of 10% SDS and sonicated to lyse the cells. After centrifugation at 15,100 × g for 5 min, 10 μL of supernatant was separated by SDS-PAGE (SuperSep Ace 10–20%, FUJIFILM Wako Pure Chemical). After electrophoresis, one gel was stained with Coomassie Brilliant Blue R250, and another gel was used to transfer proteins to FluoroTrans W membrane (PALL, New York, USA). The membrane was blocked in 5% skim milk in PBS 0.05% Tween 20 and then incubated with anti-FLAG M2 antibody HRP conjugate (1 μL in 10 ml blocking solution) (Sigma-Aldrich, St. Louis, USA) or anti-T7 antibody HRP conjugate (1 μL in 10 ml blocking solution) (Takara Bio, Shiga, Japan). Protein signals were detected using ECL Prime Western Blotting Detection Reagent (Merck, New Jersey, USA). Images were captured using FUSION FX (Vilber).
Light sensitivity of pulcherriminic acid and pulcherrimin
For the pulcherriminic acid light sensitivity assay, 100 μL pulcherriminic acid solution (OD410 = 0.4) in a 96-well plate with a cover was incubated at 30 °C under moderate light (35 μmol m−2s−1). Before and after 10, 30, and 60 min of light exposure, UV and visible light absorption spectra of the samples were recorded using a UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan). For the pulcherrimin light sensitivity assay, pulcherrimin precipitate was suspended in 650 μL water, and 100 μL of the suspension was added to the wells of a 96-well plate. Three samples were incubated at 30 °C under moderate light for 60 min, and the other three samples were incubated in the dark. The samples were then mixed with 900 μL of 2 M NaOH to convert pulcherrimin to pulcherriminic acid. After centrifugation at 15,100 × g for 3 min, the supernatant was used for OD410 measurements.
LC/PDA-MS analysis
One mg of pulcherriminic acid (Glpbio, California, USA) was dissolved in 97 μL of 5 mM NaOH. Following the neutralization of the solution to pH 7.5 with 0.6% formic acid, the aliquots of the solution were incubated under moderate light for 0, 10, or 60 min. Subsequently, the samples were acidified to pH 3 with 0.6% formic acid (the final concentration of pulcherriminic acid, 0.52 mg/mL) and subjected to LC/PDA analysis. Chromatographic separations were conducted on UHPLC “Vanquish Duo” (Thermo Fisher Scientific, San Jose, USA) with 6 µL injection, equipped with an InertSustain C18 column (100 by 2.1 mm i.d. 3 µ particle size, GL Science, Japan) at 28 °C. The mobile phase consisted of solvent A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile). The following gradient was applied at a flow rate of 200 µL min-1: 0–1 min, 100%; 1–22 min, from 100 to 0% A; 22–25 min, to 0% A, column wash; 25–30 min, to 100% A for equilibration of the column. Pulcherriminic acid was detected at 280 and 420 nm by PDA, and mass detection from m/z 250–500 by L LTQ XL™ Linear Ion Trap Mass Spectrometer (Thermo Fisher Scientific, San Jose, USA) using full scan mode covering positive (sid = 20) and negative (sid = 10; 40) ion detection. The chromatograms were analyzed by Xcalibur software version 4.1 (Thermo Fisher Scientific, San Jose, USA).
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Data availability
All data generated in this study are available within the paper and its Supplementary Information. Uncropped images and LC/PDA-MS data are available in Figshare: https://doi.org/10.6084/m9.figshare.27126231. Source data are provided with this paper as a Source Data file.
References
Davis, S. J., Vener, A. V. & Vierstra, R. D. Bacteriophytochromes: phytochrome-like photoreceptors from nonphotosynthetic eubacteria. Science 286, 2517–2520 (1999).
Swartz, T. E. et al. Blue-light-activated histidine kinases: two-component sensors in bacteria. Science 317, 1090–1093 (2007).
van der Horst, M. A., Key, J. & Hellingwerf, K. J. Photosensing in chemotrophic, non-phototrophic bacteria: let there be light sensing too. Trends Microbiol. 15, 554–562 (2007).
Gomelsky, M. & Hoff, W. D. Light helps bacteria make important lifestyle decisions. Trends Microbiol. 19, 441–448 (2011).
Wu, L., McGrane, R. S. & Beattie, G. A. Light regulation of swarming motility in Pseudomonas syringae integrates signaling pathways mediated by a bacteriophytochrome and a LOV protein. mBio 4, e00334–13 (2013).
Purcell, E. B., Siegal-Gaskins, D., Rawling, D. C., Fiebig, A. & Crosson, S. A photosensory two-component system regulates bacterial cell attachment. Proc. Natl Acad. Sci. USA 104, 18241–18246 (2007).
Bonomi, H. R. et al. Light regulates attachment, exopolysaccharide production, and nodulation in Rhizobium leguminosarum through a LOV-histidine kinase photoreceptor. Proc. Natl Acad. Sci. USA 109, 12135–12140 (2012).
Tiensuu, T., Andersson, C., Rydén, P. & Johansson, J. Cycles of light and dark co-ordinate reversible colony differentiation in Listeria monocytogenes. Mol. Microbiol. 87, 909–924 (2013).
Cai, R. et al. Blue light promotes zero-valent sulfur production in a deep-sea bacterium. EMBO J. 42, e112514 (2023).
De Luca, G. et al. Light on the cell cycle of the non-photosynthetic bacterium Ramlibacter tataouinensis. Sci. Rep. 9, 16505 (2019).
Takano, H. The regulatory mechanism underlying light-inducible production of carotenoids in nonphototrophic bacteria. Biosci. Biotechnol. Biochem. 80, 1264–1273 (2016).
Galbis-Martínez, M., Padmanabhan, S., Murillo, F. J. & Elías-Arnanz, M. CarF mediates signaling by singlet oxygen, generated via photoexcited protoporphyrin IX, in Myxococcus xanthus light-induced carotenogenesis. J. Bacteriol. 194, 1427–1436 (2012).
Tschowri, N., Busse, S. & Hengge, R. The BLUF-EAL protein YcgF acts as a direct anti-repressor in a blue-light response of Escherichia coli. Genes Dev. 23, 522–534 (2009).
Hatfield, B. M. et al. Light cues induce protective anticipation of environmental water loss in terrestrial bacteria. Proc. Natl Acad. Sci. USA 120, e2309632120 (2023).
O’Toole, G., Kaplan, H. B. & Kolter, R. Biofilm formation as microbial development. Annu. Rev. Microbiol. 54, 49–79 (2000).
Branda, S. S., González-Pastor, J. E., Ben-Yehuda, S., Losick, R. & Kolter, R. Fruiting body formation by Bacillus subtilis. Proc. Natl Acad. Sci. USA 98, 11621–11626 (2001).
Arnaouteli, S., Bamford, N. C., Stanley-Wall, N. R. & Kovács, Á T. Bacillus subtilis biofilm formation and social interactions. Nat. Rev. Microbiol. 19, 600–614 (2021).
Losi, A., Polverini, E., Quest, B. & Gärtner, W. First evidence for phototropin-related blue-light receptors in prokaryotes. Biophys. J. 82, 2627–2634 (2002).
Gaidenko, T. A., Kim, T. J., Weigel, A. L., Brody, M. S. & Price, C. W. The blue-light receptor YtvA acts in the environmental stress signaling pathway of Bacillus subtilis. J. Bacteriol. 188, 6387–6395 (2006).
Avila-Pérez, M., Hellingwerf, K. J. & Kort, R. Blue light activates the sigmaB-dependent stress response of Bacillus subtilis via YtvA. J. Bacteriol. 188, 6411–6414 (2006).
Suzuki, N., Takaya, N., Hoshino, T. & Nakamura, A. Enhancement of a sigma(B)-dependent stress response in Bacillus subtilis by light via YtvA photoreceptor. J. Gen. Appl. Microbiol. 53, 81–88 (2007).
Eelderink-Chen, Z. et al. A circadian clock in a nonphotosynthetic prokaryote. Sci. Adv. 7, eabe2086 (2021).
Sartor, F. et al. The circadian clock of the bacterium B. subtilis evokes properties of complex, multicellular circadian systems. Sci. Adv. 9, eadh1308 (2023).
Hansson, M., Rutberg, L., Schröder, I. & Hederstedt, L. The Bacillus subtilis hemAXCDBL gene cluster, which encodes enzymes of the biosynthetic pathway from glutamate to uroporphyrinogen III. J. Bacteriol. 173, 2590–2599 (1991).
Saeed, I. A. & Ashraf, S. S. Denaturation studies reveal significant differences between GFP and blue fluorescent protein. Int. J. Biol. Macromol. 45, 236–241 (2009).
Nies, D. H. & Herzberg, M. A fresh view of the cell biology of copper in enterobacteria. Mol. Microbiol. 87, 447–454 (2013).
Hamblin, M. R. et al. Helicobacter pylori accumulates photoactive porphyrins and is killed by visible light. Antimicrob. Agents Chemother. 49, 2822–2827 (2005).
Vagner, V., Dervyn, E. & Ehrlich, S. D. A vector for systematic gene inactivation in Bacillus subtilis. Microbiol. 144, 3097–3104 (1998).
Kinsinger, R. F., Shirk, M. C. & Fall, R. Rapid surface motility in Bacillus subtilis is dependent on extracellular surfactin and potassium ion. J. Bacteriol. 185, 5627–5631 (2003).
van Gestel, J., Vlamakis, H. & Kolter, R. From cell differentiation to cell collectives: Bacillus subtilis uses division of labor to migrate. PLoS Biol. 13, e1002141 (2015).
Grau, R. R. et al. A Duo of potassium-responsive histidine kinases govern the multicellular destiny of Bacillus subtilis. mBio 6, e00581 (2015).
Vlamakis, H., Aguilar, C., Losick, R. & Kolter, R. Control of cell fate by the formation of an architecturally complex bacterial community. Genes Dev. 22, 945–953 (2008).
Errington, J. Regulation of endospore formation in Bacillus subtilis. Nat. Rev. Microbiol. 1, 117–126 (2003).
Arnaouteli, S. et al. Pulcherrimin formation controls growth arrest of the Bacillus subtilis biofilm. Proc. Natl Acad. Sci. USA 116, 13553–13562 (2019).
Qin, Y. et al. Heterogeneity in respiratory electron transfer and adaptive iron utilization in a bacterial biofilm. Nat. Commun. 10, 3702 (2019).
Angelini, L. L. et al. Pulcherrimin protects Bacillus subtilis against oxidative stress during biofilm development. NPJ Biofilms Microbiomes 9, 50 (2023).
Charron-Lamoureux, V. et al. Pulcherriminic acid modulates iron availability and protects against oxidative stress during microbial interactions. Nat. Commun. 14, 2536 (2023).
Gondry, M. et al. Cyclodipeptide synthases are a family of tRNA-dependent peptide bond-forming enzymes. Nat. Chem. Biol. 5, 414–420 (2009).
Sauguet, L. et al. Cyclodipeptide synthases, a family of class-I aminoacyl-tRNA synthetase-like enzymes involved in non-ribosomal peptide synthesis. Nucleic Acids Res. 39, 4475–4489 (2011).
Cryle, M. J., Bell, S. G. & Schlichting, I. Structural and biochemical characterization of the cytochrome P450 CypX (CYP134A1) from Bacillus subtilis: a cyclo-L-leucyl-L-leucyl dipeptide oxidase. Biochem 49, 7282–7296 (2010).
Kupfer, D. G., Uffen, R. L. & Canale-Parola, E. The role of iron and molecular oxygen in pulcherrimin synthesis by bacteria. Arch. Mikrobiol. 56, 9–21 (1967).
García-Domínguez, M., Muro-Pastor, M. I., Reyes, J. C. & Florencio, F. J. Light-dependent regulation of cyanobacterial phytochrome expression. J. Bacteriol. 182, 38–44 (2000).
Braatsch, S., Moskvin, O. V., Klug, G. & Gomelsky, M. Responses of the Rhodobacter sphaeroides transcriptome to blue light under semiaerobic conditions. J. Bacteriol. 186, 7726–7735 (2004).
Tardu, M., Bulut, S. & Kavakli, I. H. MerR and ChrR mediate blue light induced photo-oxidative stress response at the transcriptional level in Vibrio cholerae. Sci. Rep. 7, 40817 (2017).
Dorey, A. L., Lee, B. H., Rotter, B. & O’Byrne, C. P. Blue Light sensing in Listeria monocytogenes is temperature-dependent and the transcriptional response to it is predominantly SigB-dependent. Front. Microbiol. 10, 2497 (2019).
Randazzo, P., Aubert-Frambourg, A., Guillot, A. & Auger, S. The MarR-like protein PchR (YvmB) regulates expression of genes involved in pulcherriminic acid biosynthesis and in the initiation of sporulation in Bacillus subtilis. BMC Microbiol. 16, 190 (2016).
Chumsakul, O. et al. Genome-wide binding profiles of the Bacillus subtilis transition state regulator AbrB and its homolog Abh reveals their interactive role in transcriptional regulation. Nucleic Acids Res. 39, 414–428 (2011).
Wang, D. et al. Regulation of the synthesis and secretion of the iron chelator cyclodipeptide pulcherriminic acid in Bacillus licheniformis. Appl. Environ. Microbiol. 84, e00262–18 (2018).
Kluyver, A. J., van der Walt, J. P. & van Triet, A. J. Pulcherrimin, the pigment of Candida Pulcherrima. Proc. Natl Acad. Sci. USA 39, 583–593 (1953).
Baichoo, N., Wang, T., Ye, R. & Helmann, J. D. Global analysis of the Bacillus subtilis Fur regulon and the iron starvation stimulon. Mol. Microbiol. 45, 1613–1629 (2002).
Quisel, J. D., Burkholder, W. F. & Grossman, A. D. In vivo effects of sporulation kinases on mutant Spo0A proteins in Bacillus subtilis. J. Bacteriol. 183, 6573–6578 (2001).
Iyanagi, T. Roles of ferredoxin-nadp+ oxidoreductase and flavodoxin in NAD(P)H-dependent electron transfer systems. Antioxidants 11, 2143 (2022).
Cybulski, L. E. et al. Mechanism of membrane fluidity optimization: isothermal control of the Bacillus subtilis acyl-lipid desaturase. Mol. Microbiol. 45, 1379–1388 (2002).
Aguilar, P. S., Hernandez-Arriaga, A. M., Cybulski, L. E., Erazo, A. C. & de Mendoza, D. Molecular basis of thermosensing: a two-component signal transduction thermometer in Bacillus subtilis. EMBO J. 20, 1681–1691 (2001).
Steingard, C. H., Pinochet-Barros, A., Wendel, B. M. & Helmann, J. D. Microbiol. Iron homeostasis in Bacillus subtilis relies on three differentially expressed efflux systems 169, 001289 (2023).
Boles, B. R. & Singh, P. K. Endogenous oxidative stress produces diversity and adaptability in biofilm communities. Proc. Natl Acad. Sci. USA 105, 12503–12508 (2008).
Mai-Prochnow, A. et al. Hydrogen peroxide linked to lysine oxidase activity facilitates biofilm differentiation and dispersal in several gram-negative bacteria. J. Bacteriol. 190, 5493–5501 (2008).
Kreth, J., Vu, H., Zhang, Y. & Herzberg, M. C. Characterization of hydrogen peroxide-induced DNA release by Streptococcus sanguinis and Streptococcus gordonii. J. Bacteriol. 191, 6281–6291 (2009).
Gozzi, K. et al. Bacillus subtilis utilizes the DNA damage response to manage multicellular development. NPJ Biofilms Microbiomes 3, 8 (2017).
Tuttobene, M. R., Cribb, P. & Mussi, M. A. BlsA integrates light and temperature signals into iron metabolism through Fur in the human pathogen Acinetobacter baumannii. Sci. Rep. 8, 7728 (2018).
Tuttobene, M. R. et al. Light modulates important pathogenic determinants and virulence in ESKAPE pathogens Acinetobacter baumannii, Pseudomonas aeruginosa, and Staphylococcus aureus. J. Bacteriol. 203, e00566–20 (2021).
Kearns, D. B. & Losick, R. Swarming motility in undomesticated Bacillus subtilis. Mol. Microbiol. 49, 581–590 (2003).
Ishihama, A. et al. Intracellular concentrations of 65 species of transcription factors with known regulatory functions in Escherichia coli. J. Bacteriol. 196, 2718–2727 (2014).
Bennett, B. D. et al. Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli. Nat. Chem. Biol. 5, 593–599 (2009).
Padmanabhan, S., Jost, M., Drennan, C. L. & Elías-Arnanz, M. A new facet of vitamin b12: gene regulation by cobalamin-based photoreceptors. Annu. Rev. Biochem. 86, 485–514 (2017).
Sipiczki, M. Metschnikowia strains isolated from botrytized grapes antagonize fungal and bacterial growth by iron depletion. Appl. Environ. Microbiol. 72, 6716–6724 (2006).
Türkel, S. & Ener, B. Isolation and characterization of new Metschnikowia pulcherrima strains as producers of the antimicrobial pigment pulcherrimin. Z. Naturforsch. C. J. Biosci. 64, 405–410 (2009).
Le Breton, Y., Mohapatra, N. P. & Haldenwang, W. G. In vivo random mutagenesis of Bacillus subtilis by use of TnYLB-1, a mariner-based transposon. Appl. Environ. Microbiol. 72, 327–333 (2006).
Ducret, A., Quardokus, E. M. & Brun, Y. V. MicrobeJ, a tool for high throughput bacterial cell detection and quantitative analysis. Nat. Microbiol. 1, 16077 (2016).
Canale-Parola, E. A red pigment produced by aerobic sporeforming bacteria. Arch. Microbiol. 46, 414–427 (1963).
Acknowledgements
This work was partly supported by the NAIST Life Science Collaboration Center (LiSCo).
Author information
Authors and Affiliations
Contributions
K.K. designed the research. K.K., R.K., and T.T. performed research and analyzed data. K.K. and T.T. wrote the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Peer review
Peer review information
Nature Communications thanks Yunrong Chai and the other, anonymous, reviewers for their contribution to the peer review of this work. A peer review file is available.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
Source data
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
About this article
Cite this article
Kobayashi, K., Kurata, R. & Tohge, T. The iron chelator pulcherriminic acid mediates the light response in Bacillus subtilis biofilms. Nat Commun 16, 5446 (2025). https://doi.org/10.1038/s41467-025-60560-4
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41467-025-60560-4
