Podospora anserina, WITH GLYCEROL

Gautier, Valérie, Nguyen, Tinh-Suong & Silar, Philippe, 2024, Fungal / bacterial syntrophy of glycerol utilization, Cryptogamie, Mycologie 20 (6), pp. 53-69 : 57-60

GROWTH OF P. ANSERINA WITH GLYCEROL AS CARBON SOURCE

As seen in Figure 2 , P. anserina appears unable to use glycerol as a sole carbon source for its vegetative growth as mycelium growth speed and density was not different on M0 medium containing 0.01 M to 0.04 M (= 0.9 g /L to 3.7 g /L) of glycerol compared to M0 medium lacking a carbon source. With higher glycerol concentrations (0.05 M to 0.3 M; i.e., 4.5 g /L to 27.6 g /L), growth of P. anserina was inhibited and even completely abolished at the highest concentrations. At 0.05 M glycerol, growth speed was inhibited by 15% (6.5 mm /day instead of 7.5 mm /day on M0 without glycerol) and at 0.1 M by 66% (2.5 mm /day instead of 7.5 mm /day). On the same media containing glycerol with concentrations up to 0.3 M, N. crassa could grow and form a profuse mycelium ( Fig. 2 ).

Interestingly, the plates containing 0.01 to 0.04 M of glycerol developed mature perithecia producing ascospores ( Fig. 2 ), while the ones lacking glycerol did not ( Fig. 2 ). These matured slowly and required three weeks to produce ripe ascospores instead of one week on the standard M2 medium. They were mainly located within the medium, were small and contained few ascospores. At higher concentrations, perithecium production was inhibited and mostly small protoperithecia were observed on the M0 medium. This indicated that P. anserina , while unable to use glycerol for its vegetative growth could use it to produce mature fruiting bodies, albeit inefficiently.

The presence of small amounts of metabolites (0.1 g /L of glucose, galactose, glutamine, peptone and yeast extract) did not improve the growth of P. anserina on M0 medium containing 0.05 M glycerol unlike what has been reported for other fungi. Indeed, the cultures with 0.05 M glycerol displayed the same growth speed, spindly mycelium morphology, and fertility as the cultures without glycerol and having only 0.1 g /L of added metabolites.

Overall, the data showed that P. anserina was unable to use glycerol as a carbon source for its vegetative growth even in the presence of metabolites, unlike what had been reported for other fungi, including its close relative N. crassa . Nevertheless, albeit inefficiently, P. anserina could use glycerol to make mature fruiting bodies.

GLYCEROL IS TOXIC TO P. ANSERINA

Growth and fertility of P. anserina on medium with glycerol as sole carbon source was inhibited at high concentrations. We thus explored whether these inhibitions were also observed on the standard M2 medium containing dextrin as a carbon source that is routinely used to grow P. anserina . As a control, we tested sorbitol at the same osmolarity as glycerol to check whether inhibition was due to high osmolarity. As seen in Figure 3 , glycerol inhibited the growth of P. anserina in M2 medium like it did in M0 medium (by 15% at 0.05 M and 69% at 0.1 M), while sorbitol did not even at the highest concentrations. We tested whether sorbitol could be used as sole carbon source and whether in these conditions high amounts of sorbitol had an inhibitory effect on P. anserina growth and fertility ( Fig. 3 ). Sorbitol was used by P. anserina as sole carbon source and, even at the highest concentration, sorbitol did not impair the growth of the fungus and was efficiently used as a sole carbon source. This indicated that glycerol was toxic to P. anserina and that toxicity was not due to an effect on osmolarity.

MINING THE GENOME OF P. ANSERINA FOR GENES INVOLVED IN GLYCEROL CATABOLISM

The P. anserina genome was mined to identify CDS possibly involved in glycerol uptake and catabolism ( Table 1 ; Fig. 1 ). We could detect by “BEST-BEST hit” using BLAST an orthologue of the S. cerevisiae STL 1 transporter. However, BLAST analyses failed to uncover homologues for S. cerevisiae FPS 1 or A. glaucus AgglpF channels. We could detect orthologues of GUT1/AN5589.2 encoding GK and GUT2/AN1396.2 encoding G3PDH (encoded by Pa_4_7610 and Pa_6_5500, respectively), indicating that P. anserina likely has a functional G3P pathway.

The presence of the DHA pathway in P. anserina needed to be clarified. Indeed, five proteins with significant levels of similarity to S. cerevisiae GCY 1 and A. nidulans AN 7193.2 NDGDs were encoded in the P. anserina genome.To identify which one was a potential NDGD, a phylogenetic tree was constructed with the five P. anserina potential NDGDs, the S. cerevisiae GCY 1 protein and its paralogue resulting from the duplication of the S. cerevisiae genome YPR1, the putative S. pombe NDGDs SPBC8 E4.04 and SPAC26F1.07, as well as all the proteins of A. nidulans similar to AN7193.2 ( Appendix 2 ). As seen in Appendix 2 , GCY1 and AN7193.2 did not appear to have true orthologues in P. anserina . However, most nodes are poorly supported owing to the small sizes of the proteins and the poor conservation of their primary sequences, making it difficult to draw a final decision as to the presence of actual NDGD in P. anserina . We tentatively propose that Pa_1_3840, Pa_1_21080, and Pa_6_7100 may be NDGDs owing to their similarity to AN7193.2 or GCY1, while no conclusion may be reached for Pa_3_2850 and Pa_3_4920. Note that a homologue for the Gld1 NDGD of S. pombe could not be found in the genomes of P. anserina , (nor in the genomes of S. cerevisiae and A. nidulans ). On the contrary, two clear homologues of the Dak1 and Dak2 DHAK of S. pombe, DAK 1 and DAK2 DHAK of S. cerevisiae , and AN0034.2 DHAK of A. nidulans were encoded in the P. anserina genome (Pa_2_11570 and Pa_6_1010).

As stated in the introduction, the key GO enzyme of the GA pathway has yet to clearly be identified in fungi. The only known enzyme endowed with a weak GO activity has been identified in P. chrysosporium ( Nguyen et al. 2018) and belongs to the GMC oxidoreductase family. The genome of P. anserina contains 28 genes encoding such enzymes, none of which has been characterized to date ( Ferrari et al. 2021). Similarly, no GDHK sequence appears available for fungi, only for mammals (https://www.brenda-enzymes.org/enzyme. php?ecno=2.7.1.28). Using sequences of GDHK from mammals, BLAST analysis identified Pa_2_11570 and Pa_6_1010 putative DHAKs as closely related to these enzymes from animals; due to the close chemical structure of dihydroxyacetone and D-glyceraldehyde ( Fig. 1 ), both may be used as substrates by Pa_2_11570 and Pa_6_1010. Finally, P. anserina has two potential ADHs encoded by Pa_6_4990 and Pa_7_1930 and one potential G3K encoded by Pa_1_23800.

Regarding a potential regulator of glycerol metabolism, we could not detect in the genome of P. anserina an obvious orthologue of the S. cerevisiae ADR 1 transcription factor involved in the regulation of GUT1 and GUT2 expression. Mining the genome of A. nidulans and N. crassa also failed to retrieve an orthologue of ADR1, suggesting that, even though A. nidulans glycerol genes are controlled by a transcription factor binding a consensus similar to the one bound by ADR1, a different transcription factor is likely involved in glycerol gene regulation in the Pezizomycotina.

We also mined the transcriptomic data generated by Silar et al. (2019), which provided an estimate of the expression of the genes in non-germinated ascospores, in ascospores eight hours after germination trigger, in 1-day-old and 4-day-old mycelia and in 2-day-old and 4-day-old perithecia ( Appendix 5). All glycerol genes were expressed at all stages of the lifecycle, including the glycerol symporter encoded by Pa_1_5200, the GK encoded by Pa_4_7610, and the G3PDH encoded by Pa_6_5500.

Overall, genome and transcriptome mining suggested that P. anserina can import glycerol and metabolize it through the G3P pathway. The presence of the DHA and GA pathways is more dubious; however, P. anserina appears to have enzymes from both pathways.

DELETION OF THE P. ANSERINA PAGUT 1 AND PAGUT 2 GENES

Because the G3P pathway appears to be the major pathway for glycerol uptake in the Pezizomycotina, including for P. anserina , we deleted the genes encoding the putative GK Pa_4_7610 and G3PDH Pa_6_5500; these were renamed PaGUT1 and PaGUT2, respectively, and the deletion mutants PaGUT1 ∆ and PaGUT2 ∆. We also retrieved the PaGUT1 ∆ PaGUT2 ∆ double mutant in the progeny of the cross between the PaGUT1 ∆ and PaGUT2 ∆ single mutants.

The mutants exhibited no phenotype on M0 and M2 media, i.e., their growth speed, mycelium morphology and fertility were indistinguishable from that of the wild type ( Fig. 4 ). On the contrary, on M0 and M2 medium in the presence of 0.05 M of glycerol, the vegetative growth of PaGUT1 ∆, PaGUT2 ∆ and PaGUT1 ∆ PaGUT2 ∆ was slightly more severely affected than that of the wild type because growth of the mutants was reduced by 20% instead of 15% at 0.05 M glycerol in the medium (speed of c. 6 mm /day instead of c. 6.5 mm /day for the wild type; see Figure 4 for pictures of the growth plates). However, at 0.1 M glycerol in M0 or M2, the growth of the mutants was as much impaired as that of the wild type ( Fig. 4 ). Importantly, unlike the wild type, PaGUT1 ∆, PaGUT2 ∆ and PaGUT1 ∆ PaGUT2 ∆ did not produce mature perithecia after three weeks incubation on medium containing 0.02 M glycerol ( Fig. 5 ). Longer incubation time did not result in the production of mature perithecia by these mutants. Finally, their fertility was like that of the wild type on an M2 medium containing glycerol. Also, on M0 and M2 media with sorbitol, the mutants grew and were fertile like the wild type.

Reintroduction of the PaGut2 wild-type allele into the mutant PaGUT2 ∆ restored a wild-type phenotype (i.e., the production of perithecia on M2+ 0.02 M glycerol). Unfortunately, we could not complement the PaGUT1 ∆ mutant because we failed to recover in bacteria a plasmid carrying the PaGut1 wild-type allele without sequence alteration (a recombination event eliminated systematically the end of the gene) and direct co-transformation with the PaGut1 wild-type allele PCR amplification product with a resistance marker yielded few transformants for an unknown reason despite several attempts; none of these had a wild-type phenotype.

Nonetheless, in crosses between PaGUT1 ∆ and the wild type the lack of perithecium production on glycerol medium co-segregated with the resistance used for deleting PaGut1, arguing that this phenotype was actually due to the deletion of PaGut1 and not to an additional unlinked mutation.

THE PRESENCE OF BACTERIA GREATLY IMPROVES THE GROWTH AND FERTILITY OF P. ANSERINA ON GLYCEROL MEDIA

We serendipitously discovered that the presence of bacteria in co-culture with P.anserina significantly improves growth and fertility of the fungus on M0 medium added with glycerol as sole carbon source. Indeed, we observed that, in a series of plates contaminated by bacteria, the bacterial colonies were surrounded by what looked like a denser hyphal thallus bearing mature perithecia ( Fig. 6 ). We identified the contaminating bacterium by sequencing its 16S barcode and found it to be Paenibacillus validus (Bacillota formerly Firmicutes).To check whether the improved growth and fertility were specific to P.validus or could be achieved by other bacteria, we tested several other species including Microbacterium proteolyticum (Actinomycetota) , Massilia sp. (Pseudomonadota – Betaproteobacteria), Paracoccus sp. (Pseudomonadota – Alphaproteobacteria), Sphingobacterium kitahiroshimense (Bacteroidota – Sphingobacteriia), Pseudomonas putida (Pseudomonadota – Gammaproteobacteria) and Escherichia coli T.Escherich DH 5α (Pseudomonadota – Gammaproteobacteria). All were found to improve the growth and fertility of P. anserina on glycerol as sole carbon source (see Figures 6 and 7 for improvement of growth and fertility of P. anserina by E. coli ).

As it is a well-known model and grows well on high glycerol concentrations ( Appendix 3 ), we chose E. coli DH 5α for further testing. We especially tested whether the growth and fertility of the PaGUT1 ∆, PaGUT2 ∆ and PaGUT1 ∆ PaGUT2 ∆ mutants were also improved in the presence of the bacteria. As seen in Figure 7 , E. coli improved the vegetative growth of all mutants and the fertility of all mutants except for PaGUT2 ∆, for which only growth was improved, as PaGUT2 ∆ did not produce perithecia in the presence of bacteria.

Figures

FIG. 2. — Growth and fertility of Podospora anserina (Rabenh.) Niessl on glycerol media. The plates were inoculated at the center with a mat+/mat- heterokaryon and incubated for three weeks at 27°C in the presence of constant light, at which time pictures were taken. Arrowheads point toward ascospore-containing perithecia.See also Figure 4 for a dark field picture of the wild-type strain growing on M0 and M0 + 0.05 M glycerol.Top, Petri plate (Ø= 8 cm).Scale bars: 200 µm.

FIG. 3. — Toxicity of glycerol. M2 and M0 Plates containing the indicated concentration of glycerol (gly) and sorbitol (sorb) were inoculated at the center with a mat+/mat- heterokaryon and incubated for two weeks at 27°C in the presence of constant light, at which time pictures were taken.Perithecia are visible as small black dots. On the M2 medium, the presence of 0.1 M of glycerol inhibited growth and abolished fertility;higher concentrations of glycerol resulted in no growth. On the contrary, Podospora anserina (Rabenh.) Niessl grew and was fertile even in M2 medium containing the highest concentration of sorbitol. Podospora anserina was also fertile on medium containing sorbitol as sole carbon source at all concentrations, while it produced no visible perithecia on a medium with the same glycerol concentrations after two weeks.

FIG. 1. — Scheme of glycerol import and catabolism in Podospora anserina (Rabenh.) Niessl as transposed from pathways known in Saccharomyces cerevisiae Meyen ex E.C.Hansen, Schizosaccharomyces pombe Lindner, and Aspergillus nidulans G.Winter.

APPENDIX 2. — Phylogenetic analysis of NDGD. The tree was rooted with the divergent protein encoded by Pa_7_11100. The AN7193 protein defined as a NDGD is in red and the Podospora anserina (Rabenh.) Niessl proteins are in blue.

FIG. 4. — Phenotype of the G3P mutants. Growth and fertility on M2 were evaluated eight days after inoculation of mat+/mat- heterokaryons of the indicated strains.For M2, the top plates are cultures on the M2 medium;the bottom ones are the cover of the same plates onto which ascospores seen as tiny black dots were expelled. Growth on M0, M0+0.05 M glycerol, and M0+ 0.1 M glycerol was evaluated after four days of growth.

FIG. 5. — The PaGut1Δ and PaGUT2Δ mutants are sterile on glycerol media. Small but mature perithecia can be seen at the periphery of the colony when the wild type is incubated for 20 days on media containing 0.02 M glycerol, while they cannot be seen in the PaGut1Δ, PaGUT2Δ, and PaGut1Δ PaGUT2Δ mutants. Bottom, enlargement of representative fruiting bodies. Scale bars: 250 µm.

FIG. 6. — Production of perithecia by Podospora anserina (Rabenh.) Niessl in the presence of Paenibacillus validus (Nakamura) Ash et al. and Escherichia coli T.Escherich. The presence of large amounts of mature perithecia (the black dots) are seen around and farther away from the bacterial colony on media containing 0.02 M glycerol. The fungus formed a dense mycelium on a plate containing 0.1 glycerol and the bacteria. Note that at 0.1 M glycerol, P. anserina remained sterile even in the presence of bacteria. Scale bars: 250 µm.

FIG. 7. — Production of mycelium and perithecia in the presence of Escherichia coli T.Escherich by Podospora anserina (Rabenh.) Niessl wild-type and mutant strains. The plates were inoculated at the same time at the center by a mat+/mat- heterokaryon of the fungus with the indicated genotypes, alone or with E. coli DH5α at the position indicated by the blue dots on the right and left of the center (visible on the M0 plates). The plates were incubated for 20 days at 27°C and constant illumination,after which they were photographed.Escherichia coli promoted the growth of the mycelium (clearly visible on plates containing 0.1 M glycerol) for all strains, and development of perithecia (clearly visible as small black dots on plates containing 0.05 M glycerol) in all strains except for the PaGut2Δ mutant.

APPENDIX 3. — Growth of Escherichia coli T.Escherich on M0 medium supplemented with glycerol. Escherichia coli DH5α was inoculated at position indicated by the blue dots (except at the one on the center where Podospora anserina (Rabenh.) Niessl would be inoculated when the plates contain the fungus, e.g. see Fig. 7) on the indicated media and the plates were incubated for 20 days at 27°C under constant illumination at which point photographs were taken. In these conditions, the growth of the E. coli colonies is readily visible after one week of incubation and starts to mask the blue dots, except on M0 that does not permit the growth of E. coli.