Bacteria Associated With Shiraia Fruiting Bodies Influence Fungal Production of Hypocrellin A

Abstract

Hypocrellin A (HA) is a natural red perylenequinone pigment from Shiraia fruiting body, which was used clinically on various skin diseases and developed as a photodynamic therapy agent against cancers. The fruiting bodies may harbor a diverse but poorly understood microbial community. In this study, we characterized the bacterial community of Shiraia fruiting body using a combination of culture-based method and Illumina high-throughput sequencing, and tested the involvement of some companion bacteria in fungal HA production using the fungal-bacterial confrontation assay. Our results revealed that the bacterial community in the fruiting body was dominated by Bacillus and Pseudomonas. Some Pseudomonas isolates such as P. fulva, P. putida, and P. parafulva could stimulate fungal HA accumulation by Shiraia sp. S9. The bacterial treatment of P. fulva SB1 up-regulated the expression of polyketide synthase (PKS) for HA biosynthesis and transporter genes including ATP-binding cassette (ABC) and major facilitator superfamily transporter (MFS) for HA exudation. After the addition of live P. fulva SB1, the mycelium cultures of Shiraia sp. S9 presented a higher HA production (225.34 mg/L), about 3.25-fold over the mono-culture. On the other hand, B. cereus was capable of alleviating fungal self-toxicity from HA via down-regulation of HA biosynthetic genes or possible biodegradation on HA. To our knowledge, this is the first report on the diversified species of bacteria associated with Shiraia fruiting bodies and the regulation roles of the companion bacteria on fungal HA biosynthesis. Furthermore, the bacterial co-culture provided a good strategy for the enhanced HA production by Shiraia.

Keywords: Shiraia fruiting body; associated bacteria; co-culture; diversity; hypocrellin A.

FIGURE 1

Scanning electron micrographes (A–G) of transverse section of Shiraia stroma. ① Perithecium; ② pseudoparenchyma; ③ prosenchyma; ④ ascus; ⑤ paraphyses; ⑥ immature ascospore; ⑦ mature ascospore; ⑧ bacterium.

FIGURE 2

Characterization of hypocrellin production by strain S9. (A) The observation of the red pigment secretion on day 10 of the strain on PDA. (B) The fermentation of the strain in PDB for 4 days. (C) The morphological structure of the strain under optical microscope (100×). ① Septal hypha; ② pycnidium; ③ conidium. (D) The typical color reaction. Pigment acetone extract (1) with 1 mol/L sodium hydroxide solution (2). Pigment acetone extract with 1 mol/L hydrochloric acid solution (3). Pigment acetone extract with 1 mol/L FeCl₃ solution (4). (E) Full wavelength scanning of the strain acetone extracts. (F) The retention time of HA of wild Shiraia sp. S9 by HPLC. (G) Qualitative analysis of HA by LC–MS. M-1, m/z [M-H] 545.4, negative ion spectra with one adduct proton. M + 1, m/z [M + H] 547.4, positive ion spectra with one adduct proton. (H) The structure of HA. Molecular formula: C₃₀H₂₆O₁₀.

FIGURE 3

Phylogenetic tree of 16S rDNA gene sequences showing the relationships between the bacteria isolated from the fruiting body and the reference strains. The branch length is proportional to the number of substitutions per site. Bar: 0.2 substitutions per nucleotide position.

FIGURE 4

Relative distribution of the bacterial genera in Shiraia fruiting body. Sequences are binned at the genus level and the abundance values are depicted as the percentage of the total bacterial sequences in a sample.

FIGURE 5

(A) The effect of live bacteria (P. fulva SB1 and B. cereus No. 1) on the growth and red pigment secretion of Shiraia sp. S9. The blank control group without treatment (a and b). The treatment group treated with live SB1 cells (c and d) and No. 1 cells (e and f). (B) The retention time of HA of Shiraia sp. S9. (C) The HA content of Shiraia sp. S9 in PDA plate. A small piece (5 mm × 5 mm) of the strain was placed in the center of a 10-cm PDA plate at 28°C for 4 days. The single colony of bacterium was inoculated in LB at 37°C on a rotary shaker at 200 rpm for 12 h. Then, bacterial suspension (10 μl) was streaked in two parallel straight lines on PDA at 28°C for 10 days, approximately 7 cm apart from each other. Values are mean ± SD from three independent experiments. Different letters above the bars mean significant differences (p < 0.05).

FIGURE 6

(A) Time profiles of growth and HA accumulation in submerged culture of S9 strain. (B) Observation of submerged cultures of Shiraia sp. S9 with or without live bacterial cells on day 8. (a) Control group. (b) Live P. fulva SB1 treatment. (c) B. cereus No. 1 treatment. (d) The effect of live bacteria on hyphal biomass, intracellular HA, extracellular HA, and total HA production in submerged cultures of Shiraia sp. S9. The bacteria at 100 cells/ml were added to the cultures on day 4. Values are mean ± SD from three independent experiments. ^∗p < 0.05, ^∗∗p < 0.01 versus control group.

FIGURE 7

The effect of live bacteria on the expression of Shiraia HA biosynthetic genes. The bacteria SB1 and No. 1 at 100 cells/ml were added to the cultures on day 4 for 24 h, respectively. PKS, polyketide synthase; Omef, O-methyltransferase; Mono, monooxygenase; MCO, multicopper oxidase; FAD, FAD/FMN-dependent oxidoreductase; ZFIF, zinc finger transcription factor; MFS, major facilitator superfamily; ABC, ATP-binding cassette transporter. Values are mean ± SD from three independent experiments. ^∗p < 0.05, ^∗∗p < 0.01 versus control group.

FIGURE 8

Effect of live SB1 treatment at different concentrations on mycelium dry biomass (A), HA content in mycelium (B), the released HA in cultural broth (C), and total HA production (D) in submerged culture of Shiraia sp. S9. The culture was maintained in a 150-ml flask containing 50 ml of the liquid medium at 150 rpm and 28°C for 8 days and treated by live SB1 on day 4. The control represents the condition with no live SB1 treatment. Values are mean ± SD from three independent experiments. Different letters above the bars mean significant differences (p < 0.05).

FIGURE 9

Effect of the addition time of live SB1 on mycelium dry biomass (A), HA content in mycelium (B), the released HA in cultural broth (C), and total HA production (D) in submerged culture of Shiraia sp. S9. The Shiraia culture was maintained in a 150-ml flask containing 50 ml of the liquid medium at 150 rpm and 28°C for 8 days. Live SB1 was added in the Shiraia cultures on day 0–7 at 400 cells/ml. The control represents the condition with no live SB1 treatment. Values are mean ± SD from three independent experiments. Different letters above the bars mean significant differences (p < 0.05).

FIGURE 10

Time profiles of mycelium dry biomass (A), HA content in mycelium (B), the released HA in cultural broth (C), and total HA production (D) in submerged culture of Shiraia sp. S9 with or without live SB1 treatment at 400 cells/ml on day 6. The culture was maintained in a 150-ml flask containing 50 ml of the liquid medium at 150 rpm and 28°C for 8 days. The arrow represents the time of addition of live SB1. Values are mean ± SD from three independent experiments. ^∗p < 0.05 and ^∗∗p < 0.01 versus control.

FIGURE 11

The bacterial susceptibilities to HA. P. fulva SB1 (A) or B. cereus No. 1 (B) was incubated in LB with HA at 0–100 mg/L on a rotary shaker at 150 rpm and 28°C for 24 h in the dark. (C) The toxicity of HA on the fungal growth of Shiraia sp. S9. HA at different concentrations (0.0–2.2 mg/L) was added to the fungal spore suspension (800 μl of 1 × 10⁵ spores/ml) with or without addition of No. 1 cells for 8 days. (D) Time profiles of HA degradation in LB with No. 1 cells. The bacterial cells (0.75 × 10³ cells/ml, 400 μl) mixed with 60 mg/L HA were incubated in the dark on a rotary shaker at 150 rpm and 28°C. MIC, minimum inhibitory concentration. Values are mean ± SD from three independent experiments. ^∗p < 0.05, ^∗∗p < 0.01 versus control group.