The CBS/H2S signalling pathway regulated by the carbon repressor CreA promotes cellulose utilization in Ganoderma lucidum

Abstract

Cellulose is an important abundant renewable resource on Earth, and the microbial cellulose utilization mechanism has attracted extensive attention. Recently, some signalling molecules have been found to regulate cellulose utilization and the discovery of underlying signals has recently attracted extensive attention. In this paper, we found that the hydrogen sulfide (H₂S) concentration under cellulose culture condition increased to approximately 2.3-fold compared with that under glucose culture condition in Ganoderma lucidum. Further evidence shown that cellulase activities of G. lucidum were improved by 18.2-27.6% through increasing H₂S concentration. Then, we observed that the carbon repressor CreA inhibited H₂S biosynthesis in G. lucidum by binding to the promoter of cbs, a key gene for H₂S biosynthesis, at "CTGGGG". In our study, we reported for the first time that H₂S increased the cellulose utilization in G. lucidum, and analyzed the mechanism of H₂S biosynthesis induced by cellulose. This study not only enriches the understanding of the microbial cellulose utilization mechanism but also provides a reference for the analysis of the physiological function of H₂S signals.

Fig. 1. Biosynthesis of H₂S was improved under cellulose culture condition.

a, b Change in the H₂S concentration was measured by SF7-AM fluorescence probe in wild-type (wt) strain under glucose or cellulose culture condition. The average fluorescence intensity values of all mycelia in the 6 photos were quantified. Scale bar = 100 μm. c Log₂(foldchange) of genes expression level of putative H₂S biosynthetic enzymes: L-cysteine desulfhydrase (lcd1 and lcd2), cysteine synthase (cs1 and cs2), cystathionine γ-lyase (cse1, cse2, cse3 and cse4), cystathionine β-synthase (cbs), 3-mercaptopyruvate sulfurtransferase (3-mst). The different letters indicate significant differences between the lines (“*“ means p < 0.05, “**“ means p < 0.01, “***“ means p < 0.001, according to Student’s t test). d, e Change in the H₂S concentration was measured by SF7-AM fluorescence staining in wt, sicontrol, cbs-silenced and cbs-overexpressed strains under glucose and cellulose culture condition. Scale bar = 100 μm. f Relative expression level of cbs gene in wt, sicontrol, cbs-silenced and cbs-overexpressed strains, cultured under glucose and cellulose condition. The different letters indicate significant differences between the lines (p < 0.05, according to Duncan’s multiple range test).

Fig. 2. H₂S enhances endocellulase activity.

a, b Endocellulase (CMCase) activities in wt strain under cellulose culture condition in the presence of sodium hydrosulfide (NaHS, H₂S donor) and hypotaurine (HT, H₂S scavenger). c, d Endocellulase (CMCase) activities in wt, sicontrol, cbs-silenced and cbs-overexpressed strains under cellulose culture condition in the in presence of NaHS and HT. e, f The growth length of wt, sicontrol, cbs-silenced and cbs-overexpressed strains after 15 days cultivated on wood chips. The red line marked the position of the wt strain grown on wood chips for 15 days. The different letters indicate significant differences between the lines (“**“ means p < 0.01, “***“ means p < 0.001, according to Student’s t test).

Fig. 3. CreA bound to cbs promoter at “CTGGGG”.

a The result of highly conservative transcription factors (TFs) selected by a yeast one-hybrid (Y1H) library screen (n = 3). b Diagram of cbs promoter sequence. The black boxes indicate the binding sequence of CreA. c Y1H assay verification between CreA and cbs promoter. d Electrophoretic mobility shift assay (EMSA) for binding action of purified CreA or nuclear extraction with cbs promoter.

Fig. 4. CreA inhibits cbs transcription and reduces H₂S biosynthesis.

a, b Immunoblot analysis of CreA proteins in wt, sicontrol, creA-silenced and creA-overexpressed strains, cultured on CYM medium for 7 days. c Relative expression level of cbs gene in wt, sicontrol, creA-silenced and creA-overexpressed strains, cultured on CYM medium for 7 days. d, e Change in the H₂S concentration was measured by SF7-AM fluorescence staining in wt, sicontrol, creA-silenced and creA-overexpressed strains, cultured on CYM medium for 7 days, measured by SF7-AM. Scale bar = 100 μm. The different letters indicate significant differences between the lines (“**“ means p < 0.01, “***“ means p < 0.001, according to Student’s t test).

Fig. 5. Cellulose culture condition activates CBS/H₂S signal pathway by reducing CreA binding to the cbs promoter.

a ChIP assays for relative binding action with cbs promoter in wt, sicontrol, creA-silenced and creA-overexpressed strains, cultured under glucose and cellulose condition. b Relative expression level of cbs gene in wt, sicontrol, creA-silenced, creA-overexpressed and creA-cbs-silenced strains, cultured under glucose and cellulose condition. c, d Change in the H₂S concentration was measured by SF7-AM fluorescence staining in wt, sicontrol, creA-silenced, creA-overexpressed and creA-cbs-silenced strains, cultured under glucose and cellulose condition. Scale bar = 100 μm. The different letters indicate significant differences between the lines (p < 0.05, according to Duncan’s multiple range test).

Fig. 6. CreA inhibits the positive regulation of CBS/H₂S in cellulose utilization of G. lucidum.

a CMCase activities in wt, sicontrol, creA-silenced, creA-overexpressed, creA-cbs-silenced and cbs-silenced strains under cellulose culture condition in the presence of NaHS. b, c The growth length of wt, sicontrol, creA-silenced, creA-overexpressed and creA-cbs-silenced strains after 15 days cultivated on wood chips. The red line marked the position of the wt strain grown on wood chips for 15 days. The different letters indicate significant differences between the lines (“*“ means p < 0.05, “**“ means p < 0.01, “***“ means p < 0.001, according to Student’s t test).

Fig. 7. Schematic representation.

The binding of CreA and cbs promoter reduced, and the transcription level of cbs gene increased, H₂S biosynthesis promoted under cellulose culture condition. H₂S improve the cellulase activity and cellulose utilization of G. lucidum.