Light Signaling Regulates Aspergillus niger Biofilm Formation by Affecting Melanin and Extracellular Polysaccharide Biosynthesis

Abstract

Light is an important signal source in nature, which regulates the physiological cycle, morphogenetic pathways, and secondary metabolites of fungi. As an external pressure on Aspergillus niger, light signaling transmits stress signals into the cell via the mitogen-activated protein kinase (MAPK) signaling pathway. Studying the effect of light on the biofilm of A. niger will provide a theoretical basis for light in the cultivation of filamentous fungi and industrial applications. Here, the characterization of A. niger biofilm under different light intensities confirmed the effects of light signaling. Our results indicated that A. niger intensely accumulated protective mycelial melanin under light illumination. We also discovered that the RlmA transcription factor in the MAPK signaling pathway is activated by light signaling to promote the synthesis of melanin, chitin, and other exopolysaccharides. However, the importance of melanin to A. niger biofilm is rarely reported; therefore, we knocked out key genes of the melanin biosynthetic pathway-Abr1 and Ayg1 Changes in hydrophobicity and electrostatic forces resulted in the decrease of biofilm caused by the decrease of melanin in mutants.IMPORTANCE As an important industrial filamentous fungus, Aspergillus niger can perceive light. The link between light signaling and A. niger biofilm is worthy of further study since reports are lacking in this area. This study found that light signaling promotes biofilm production in A. niger, wherein melanin plays an important role. It was further discovered that the RlmA transcription factor in the mitogen-activated protein kinase (MAPK) signaling pathway was mediated by light signaling to promote the synthesis of melanin and extracellular polysaccharides. These findings set the stage for light signal regulation of biofilm in filamentous fungi and provide a theoretical basis for the development of a new light-controlled biofilm method to improve biofilm-based industrial fermentation.

Keywords: Aspergillus niger; MAPK signaling pathway; biofilm; light signaling; melanin.

FIG 1

Effect of light on biofilm formation in A. niger. (A) Various amounts of A. niger wild-type spores were inoculated into a 24-well plate, incubated at 30°C in the dark, exposed to light intensity of 1,000 to 4,000 lx for 36 h, and then photographed after CV staining. (B) The corresponding OD₅₇₀ value in the 24-well plate. The values represent the means and standard deviations of three independent experiments. ***, P < 0.001; **, P < 0.01; *, P < 0.05; two-way ANOVA.

FIG 2

Appearance and microscopic morphology of A. niger grown in light or darkness. (A) Biofilm on round coverslip after light or dark culture; image was taken by Nova NanoSEM. Scale bar, 40 μm. (B) Biofilm on round coverslip after light or dark culture; image taken by cryo-SEM. Scale bar, 50 μm. (C) A. niger colony growing on PDA solid medium under light or dark conditions. (D) Different colors of A. niger mycelium collected under light or dark conditions (YPD liquid medium). (E) Microscope graphics of A. niger hyphae in YPD liquid medium under light or dark conditions. Scale bar, 200 μm.

FIG 3

Impact of light sensing on ECM. (A) Immunofluorescence staining images of A. niger biofilm mycelium and polysaccharides taken with a confocal laser-scanning microscope under light or dark conditions. Red indicates PI-stained DNA, and green indicates FITC-ConA-stained exopolysaccharides. Scale bar, 800 μm by 800 μm and 100 μm. (B) The content of β-1,3-glucan, chitin, and α-1,3-glucan. The values represent the means and standard deviations of three independent experiments. ***, P < 0.001; **, P < 0.01; *, P < 0.05; using the Student’s t test.

FIG 4

Elevated expression of related genes and proteins under light. (A) Expression of genes involved in the synthesis of chitin, GAG, GM, α-1,3-glucan, and β-1,3-glucan. (B) Expression of genes in melanin biosynthesis. (C) Expression of key genes in the MAPK signaling pathway. (D) Western blot results of Hog1 and MpkA protein expression and phosphorylation level under light and dark conditions. (E) Relative abundance of MpkAp and Hog1p and phosphorylated MAPK ratio under light or dark conditions. P-p42/p42 and P-α-Hog1/α-Hog1 represented the ratio of phosphorylated proteins to total proteins, calculated by means. The values represent the means and standard deviations of three independent experiments. ***, P < 0.001; **, P < 0.01; *, P < 0.05; using the Student’s t test.

FIG 5

Light promotes the production of melanin. (A) Microscope images of A. niger under light or dark conditions. Scale bar, 200 μm and 50 μm. (B) The content of melanin. The values represent the means and standard deviations of three independent experiments. ***, P < 0.001; **, P < 0.01; *, P < 0.05; using the Student’s t test.

FIG 6

Characterization of hydrophobicity and electrostatic force of A. niger ΔAbr1 and ΔAyg1 strains. (A) Growth of ΔAbr1 and ΔAyg1 strains on normal plates. (B) RNA level of RodA in ΔAbr1 and ΔAyg1 strains. (C) Zeta potential of ΔAbr1 and ΔAyg1 spores. (D) Hydrophobicity detection of ΔAbr1 and ΔAyg1 spores. The values represent the means and standard deviations of three independent experiments. ***, P < 0.001; **, P < 0.01; *, P < 0.05; using the Student’s t test.

FIG 7

Biofilm formation ability and resistance to cell wall disrupters in A. niger ΔAbr1 and ΔAyg1 strains. (A and B) Image and OD₅₇₀ of A. niger ΔAbr1 and ΔAyg1 strain CV assay results. (C) resistance of A. niger ΔAbr1 and ΔAyg1 strains to Congo red and Calcofluor white. (D) SEM images of A. niger ΔAbr1 and ΔAyg1 strains. Scale bar, 50 μm. The values represent the means and standard deviations of three independent experiments. ***, P < 0.001; **, P < 0.01; *, P < 0.05; using the Student’s t test.

FIG 8

Schematic diagram of light signaling-mediated MAPK signaling pathway involved in A. niger biofilm formation. Solid arrow, activation; dotted arrow, indirect effect.