Trehalose synthesis in Aspergillus niger: characterization of six homologous genes, all with conserved orthologs in related species
© Svanström et al.; licensee BioMed Central Ltd. 2014
Received: 23 December 2013
Accepted: 8 April 2014
Published: 11 April 2014
The disaccharide trehalose is a major component of fungal spores and is released upon germination. Moreover, the sugar is well known for is protective functions, e.g. against thermal stress and dehydration. The properties and synthesis of trehalose have been well investigated in the bakers’ yeast Saccharomyces cerevisiae. In filamentous fungi, such knowledge is limited, although several gene products have been identified.
Using Aspergillus niger as a model fungus, the aim of this study was to provide an overview of all genes involved in trehalose synthesis. This fungus has three potential trehalose-6-phosphate synthase encoding genes, tpsA-C, and three putative trehalose phosphate phosphatase encoding genes, tppA-C, of which two have not previously been identified. Expression of all six genes was confirmed using real-time PCR, and conserved orthologs could be identified in related Aspergilli. Using a two-hybrid approach, there is a strong indication that four of the proteins physically interact, as has previously been shown in S. cerevisiae. When creating null mutants of all the six genes, three of them, ΔtpsA, ΔtppA and ΔtppB, had lower internal trehalose contents. The only mutant with a pronounced morphological difference was ΔtppA, in which sporulation was severely reduced with abnormal conidiophores. This was also the only mutant with accumulated levels of trehalose-6-phosphate, indicating that the encoded protein is the main phosphatase under normal conditions. Besides ΔtppA, the most studied deletion mutant in this work was ΔtppB. This gene encodes a protein conserved in filamentous Ascomycota. The ΔtppB mutant displayed a low, but not depleted, internal trehalose content, and conidia were more susceptible to thermal stress.
A. niger contains at least 6 genes putatively involved in trehalose synthesis. Gene expressions related to germination have been quantified and deletion mutants characterized: Mutants lacking tpsA, tppA or tppB have reduced internal trehalose contents. Furthermore, tppA, under normal conditions, encodes the functional trehalose-6-phosphate-phosphatase.
Trehalose (α-D-glucopyranosyl-α-D-glucopyranoside) is a non-reducing disaccharide that is present in a wide variety of organisms. It has been isolated from plants, fungi, nematodes and insects [1–3]. In fungi, trehalose has been shown to accumulate in dispersal and survival structures such as spores (where it can constitute as much as 10% of the dry weight), sclerotia, and in yeast cells going into stationary phase [3, 4] . Since the sugar is rapidly degraded when these structures germinate or resume vegetative growth, early research concluded that trehalose serves as a storage molecule [5, 6]. However, later studies showed that the function of trehalose is more complex and diverse than just serving as an energy reserve; the molecule has been shown to function as a regulator of carbon metabolism , a signaling molecule and a protection molecule against various kinds of abiotic stress [3, 7]. Several fungal species have been shown to induce trehalose production as a stress response. Examples include: Saccharomyces cerevisiae[8, 9], Zygosaccharomyces bailii, A. nidulans, A. fumigatus, Rhizopus oryzae, and Botrytis cinerea. Trehalose is known to protect both proteins and lipid membranes of living cells against stressors such as heat, desiccation and cold. Although the mode of bio-protection of trehalose is not fully elucidated, three main hypotheses are generally accepted, and the true mechanism is likely a combination of these. The hypotheses include: water replacement (direct interaction of trehalose with the protected structure through hydrogen bonds); mechanical entrapment (glass formation of trehalose that creates a protective coating around the structure); preferential exclusion (bulk water is ordered around trehalose and is thereby separated from the bio-molecule, which then becomes more compact and stabilized) [15, 16]. The physico-chemical properties of trehalose that lie behind these hypotheses include several crystalline forms, a high glass transition temperature, and the stereochemistry of the sugar [7, 15].
In fungi, trehalose is synthesized via the intermediate trehalose-6-phosphate (T6P) and involves two enzymatic steps. First, T6P is formed from one glucose-6-phosphate and one UDP-glucose catalyzed by T6P-synthase (here called TPS). In the next step, the phosphate molecule is removed by trehalose-phosphate-phosphatase (here called TPP) yielding trehalose [1, 11]. The organism in which trehalose synthesis has been most thoroughly studied is S. cerevisiae. Here, four homologous gene products responsible for trehalose synthesis physically interact forming a “trehalose synthase complex”, which consists of one TPS (called Tps1), one TPP (called Tps2), and two other subunits, Tsl1 and Tps3, with proposed regulatory and stabilizing functions [6, 17–19]. In filamentous fungi, the gene products involved in trehalose synthesis are not as thoroughly investigated as in S. cerevisiae, but have been studied with respect to germination , plant pathology  and human pathology [12, 22].
Within Aspergilli, several individual gene products have been identified and characterized. In A. niger, two Tps1 orthologs, tpsA and tpsB, have been identified and characterized. At ambient temperature, the trehalose level of ΔtpsA mycelia was lowered compared to wild-type. In contrast to the constitutively expressed tpsA, the expression of tpsB was induced by thermal stress . In the opportunistic human pathogen A. fumigatus, four Tps1 paralogs, tpsA – D have been identified . When deleting these genes, the authors found that either tpsA or tpsB was sufficient to maintain normal trehalose levels, but if both genes were deleted, the resulting mutant strain was depleted of trehalose and showed slower germination rates as well as higher susceptibility to heat and oxidative stress compared to wild-type. Another notable finding was that this double mutant was hypervirulent in infected mice . In A. nidulans, a Tps1 ortholog, tpsA, has been identified and deleted. In this mutant, trehalose was not accumulated, and in addition, the authors could conclude that in A. nidulans trehalose is important for resistance to continual exposure to sub-lethal stress but not to short exposure of lethal stress . In contrast to S. cerevisiae, tps mutants in Aspergilli are able to utilize glucose as carbon source [11, 23, 24]. All identified Tps1 orthologs in Aspergilli are generally much shorter than the S. cerevisiae Tps1, around 500 amino acids compared to 1447.
Besides Tps1 orthologs, two Tps2 orthologs have been identified within the Aspergilli, one in A. nidulans and one in A. fumigatus: In both species they are designated orlA. The ΔorlA mutant of A. fumigatus had a pronounced phenotype with abolished asexual reproduction as well as decreased virulence. However, the phenotype could be restored to wild-type appearance by growing the mutant on media containing an osmotic stabilizer (sorbitol or glycerol). As also observed in A. nidulans, the A. fumigatus ΔorlA mutant strain contained wild-type levels of trehalose but the T6P levels were elevated [22, 25].
In this study we focused on trehalose synthesis in filamentous fungi, and more specifically, in Aspergillus niger. This is a common food spoilage mould as well as an industrially important organism, utilized for production of citric acid, for instance . Six genes, tpsA (ANI_1_1406074), tpsB (ANI_1_1078064), tpsC (ANI_1_1216124), tppA (ANI_1_1432094), tppB (ANI_1_48114) and tppC (ANI_1_2070064) were identified to be involved in trehalose biosynthesis. Expression of these genes was studied during conidial outgrowth. In addition, we deleted these genes and characterized the mutants in terms of trehalose and T6P content, protein interactions, and stress survival coupled to situations often occurring in foodstuff.
Software, hardware and computer-based analyses used in this study
GraphPad Prism® version 5 was used for generating figures (line drawings) and calculating mean, standard error of the mean, and significance between samples (using one or two way ANOVA and Bonferroni post-test). Adobe Illustrator CS5 and Adobe Photoshop CS6 were used for managing pictures (cropping and minor changes in contrast levels for best visualization). Bio-Rad CFX 96™ Real-Time System was used for generating gene expression data and the Bio-Rad CFX Manager™ version 1.6 software was used for analyzing the data. MacVector version 12 was used for primer design and phylogenetic analyses.
Culture maintenance, spore preparation and spore densities
Strains used in this study
cspA1,pyrG1, ∆kusA::amdS +
cspA1,pyrG1, ∆kusA::DR-amdS + -DR
cspA1,pyrG1, ∆kusA::amdS + , ∆tpsA::pyrG
cspA1,pyrG1, ∆kusA::amdS + , ∆tpsB::pyrG
cspA1,pyrG1, ∆kusA::amdS + , ∆tppB::pyrG
cspA1,pyrG1, ∆kusA::amdS + , ∆tppB::pyrG
cspA1,pyrG1, ∆kusA::amdS + , ∆tppC::pyrG
cspA1, ∆kusA::amdS +
cspA1,pyrG1, ∆tppB::pyrG, tppB::hph
Low-temperature scanning electron microscopy (SEM)
Wild-type, N402, and ΔtppA were grown for 1 week on AMM. Margins of colonies containing conidiophores were excised with a surgical blade and carefully transferred into a copper cup (diameter 10 mm, height 8 mm). Dislodging during snap freezing was prevented by gluing agar blocks in the copper cup with frozen tissue medium (KP-Cryoblock, Klinipath, Duiven, the Netherlands). The sample was snap-frozen in nitrogen slurry and immediately transferred in a vacuum transfer device to an Oxford CT1500 Cryostation attached to a JEOL 5600LV scanning electron microscope (JEOL, Tokyo, Japan). Electron micrographs were acquired from uncoated frozen samples, or after sputter-coating with gold three times during 30 s. Micrographs of uncoated samples were taken at an acceleration voltage of 2.5 kV, and consisted of 30 averaged fast scans (SCAN 2 mode). Coated samples were observed at 5 kV using F4 scans.
Extraction of nucleic acids
DNA was extracted as previously described . RNA from dormant conidia and conidia in early stages of germination (0 and 3 h) was extracted according to Leeuwen and co-workers . RNA from germinating spores (6 and 12 h), mycelia and sporulating mycelia (plate) were extracted according to Plumridge and co-workers . As a final step in both protocols, the RNA products were purified using a Qiagen RNeasy Mini kit (RNA clean up protocol).
Primers used for cDNA synthesis, qPCR and Two-Hybrid cloning
Confirmation of cloned cDNA to pKT25 vector
Confirmation of cloned cDNA to pUT18C vector
Cloning of tpsA cDNA
Cloning of tpsB cDNA
Cloning of tpsC cDNA
Cloning of tppA cDNA
Cloning of tppB cDNA
Cloning of tppC cDNA
cDNA synthesis and Real-Time PCR
Using total RNA as template, cDNA was synthesized using a polyT primer (Table 2) and the enzyme SuperscriptIII (Invitrogen) according to the manufacturer’s protocol. Quantitative real-time PCR was performed with the BioRad CFX-96 system using the EvaGreen reagent (BioRad), gene specific primers (Table 2), and the following protocol: Initial denaturation and enzyme activation, 95°C 30 s; 40 cycles of 95°C for 2 s and 56-60°C for 8 s; plate read; and finally, melt curve analysis starting at 65°C and ending at 95°C. Relative expression for tpsA-C and tppA-C were calculated from and compared to a serially-diluted cDNA pool and normalized to the actin-encoding gene (ANI_1_106134), which has been successfully used in previous experiments [28, 31] and is expressed at high levels throughout germination according to published microarray data . For each growth stage, the expressions were calculated from four biological replicates, each with three technical replicates. To verify the expression, or lack thereof, in the reconstituted and null mutant of tppB, the expression in mutants was normalized against N402 as previously described  using the efficiency calibrated mathematical method for the relative expression ratio in real-time PCR .
Gene deletions and complementation
Primers used for targeted gene deletions
Amplifies pyrG with 3' tpsA overhangs
tpsA, upstream fragment
tpsA, downstream fragment
Amplification of KO-fragment
Amplifies pyrG with 3′ tpsB overhangs
tpsB, upstream fragment
tpsB, downstream fragment
Amplification of tpsB KO-fragment
pyrG, KO of tpsC
Cloning of tpsC
Amplification of tpsC KO-fragment
pyrG with 3′ tppA overhangs
tppA, upstream fragment
tppA, downstream fragment
Amplification of tppA KO-fragment
Amplification of A. oryzae pyrG with 3′ tppB overhangs
tppB, upstream fragment
tppB, downstream fragment
Amplification of tppB KO-fragment
pyrG with 3′ tppC overhangs
tppC, upstream fragment
tppC, downstream fragment
Amplification of tppC KO-fragment
Amplification of tpsB/tppC double mutant
Extraction and quantification of trehalose and trehalose-6-phosphate
Trehalose from dormant and swollen conidia, germlings and mycelia was extracted and quantified as previously described . In brief, harvested fungal material was freeze-dried and homogenized using a mortar. Samples were diluted with ultra pure water, boiled, evaporated and derivatized by trimethylsilylanization before injection into the gas chromatograph–mass spectrometer (GC–MS). Relative concentrations of α-α-trehalose were calculated as the ratio to an internal standard (α-β-trehalose) and thereafter correlated to a standard curve to obtain the absolute concentrations. All trehalose measurements were performed in biological duplicates based on the average of three technical triplicates.
Extraction and quantification of T6P was performed essentially as described by . Liquid cultures were inoculated with 106 spores per ml, incubated at 25°C for 3 days at 140 rpm, and all mycelia from one culture made up one sample. Three biological replicates based on the average of three technical replicates were used for all strains.
Stress tolerance and long term viability of conidia
Dormant conidia from wild-type A. niger, the additional control strain pyrG+, and the deletion mutants ΔtppB and ΔtppB 2 were subjected to heat stress for 20, 60, 90 and 120 min at 55°C. Dormant conidia of wild-type, pyrG + and ΔtppB were subjected to sub-lethal salt and benzoic acid stress by being spread on AMM plates containing benzoic acid or NaCl at concentrations ranging from non-effective to total growth inhibition of the control strains. For detailed description of these stress experiments see . In addition, dormant conidia from control strains and ΔtppB were subjected to oxidative stress by adding 200 mM H2O2 to freshly made conidial suspensions (approximately 250 spores/ml liquid AMM). The suspensions were incubated for 10, 20 or 40 min before being spread on AMM plates. To test long-term viability, conidial suspensions (106 conidia/ml water) were stored at 4°C for a total of 8 weeks. An aliquot of the suspension was withdrawn weekly, diluted and spread on AMM plates for enumeration.
Plates from all experiments were incubated at 25°C for 3–7 days before CFU were estimated, and all experiments were performed at least in triplicates (based on three technical replicates).
Identification of genes involved in trehalose synthesis in Aspergillus niger and other fungi
Two-hybrid assay to reveal putative protein-protein interactions
Protein-protein interactions assayed by Bacterial adenylate cyclase two-hybrid system
Gene expression during conidial outgrowth
The general expression pattern of the genes (Figure 3) was as follows: The expression was highest in still dormant conidia and had decreased by approximately 2-fold after 3 h incubation; after 6 h incubation there was a slight, but not significant, decrease; and, in 12 and 72 h mycelium the expression was very low. For tpsB, tppA and tppC, the expression was then up-regulated in sporulating colonies (5 days old), while it remained low for tpsC and tppB. One gene, tppA, deviated slightly from the described pattern: The decrease in expression after 3 h was not as profound as in the other genes, and a slight, but not significant, up-regulation could be seen in 72 h mycelium.
Targeted gene deletions of six Aspergillus niger genes
To characterize the function of the six A. niger proteins, tpsA, tpsB, tpsC, tppA, tppB and tppC were all subjected to targeted gene deletions by replacing the gene with the A. oryzae pyrG resistance cassette. A double mutant, lacking the two adjacent genes tpsB and tpsC was also constructed. All deletion mutants were confirmed with PCR using both internal and flanking primers (data not shown).
Quantification of trehalose-6-phosphate and trehalose in wild-type and mutants
At all time points, conidia from all mutant strains contained significantly less trehalose compared to wild-type conidia (again, with the exception of ΔtpsB-ΔtppC 28 days). When comparing the deletion mutants to the other control strain, pyrG+, significantly lower levels of trehalose were detected in strains ΔtpsA, ΔtppA and ΔtppB. After 14 days of maturation the conidial trehalose level was 50% lower in ΔtpsA compared to pyrG+, and 73 and 60% lower in ΔtppA and ΔtppB, respectively. For ΔtpsA and ΔtppA, the reduction was significant at all time points tested, and for ΔtppB, the difference was significant in 14, 28 and 90 day old conidia but not after 5 days.
Among the deletion mutants with wild-type like phenotypes, i.e. when excluding ΔtppA, ΔtppB had the lowest overall trehalose content. After 14 days of incubation, the trehalose level was 1.7% of conidial dry weight compared to 5.1 and 4.1% in wild-type N402 and pyrG+, respectively. Although the conidial trehalose content was consistently lower in ΔtppA, the extremely low number of spores produced made this strain unsuitable for studies on conidial survival. Therefore, ΔtppB was, due to its wild-type morphology, selected for additional studies to reveal whether or not a normal internal trehalose level has any impact on stress survival and growth.
Confirmation and further characterization of ΔtppB
In this project we have studied six genes with a putative role in trehalose synthesis in A. niger: tpsA, tpsB, tpsC, tppA, tppB and tppC. All six genes encode homologous proteins and no similar gene products within the A. niger genome could be detected. Three proteins, TpsA, TpsB and TpsC, have previously been identified as orthologs to the yeast protein Tps1. As the orthologs are conserved in related species, it is plausible that there is a functional differentiation between the paralogs, e.g. one paralog could be essential for trehalose synthesis in conidia, whereas another paralog is strictly induced by stress. This assumption is in line with the previous observation in A. niger where the expression of tpsB is stress-induced whereas tpsA is constitutively expressed , although our data also suggest that tpsB has a role during differentiation (see Figure 3). When deleting the trehalose-phosphate-synthase paralogs, only ΔtpsA displayed a reduced trehalose content. The lower level in this mutant is in line with a previous report using a different target strain and deletion procedure . In the related fungus, A. fumigatus, a tpsA/tpsB double deletion resulted in a strain with depleted trehalose content, and in the same study, it was shown that the expressions of tpsC and –D were very low at all time points . These authors evaluated their expression data using RNA extracted from hyphae, and in the present study, the A. niger tpsC was expressed at very low levels at 72 h. Thus the results from the two fungi are not contradictory, and most likely an A. niger tpsA/tpsB deletion mutant would also have a depleted trehalose content. The results from A. niger and A. fumigatus are also in accordance with findings in A. nidulans where deletion of tpsA resulted in depleted trehalose content , as that species does not have the tpsB paralogue. A conclusion from studying the trehalose content from these three species is that TpsA is the most important trehalose-phosphate-synthase under normal conditions, but lack of the tpsA gene can be fully compensated by TpsB in A. fumigatus and partly by at least one of TpsB or TpsC in A. niger, but not by TpsD in A. nidulans.
The deletion mutant with the most distinctive characteristics in our experiments was ΔtppA, i.e. with an abnormal morphology and reduced levels of both trehalose-6-phosphate and trehalose. The altered morphology of the strain is probably due to toxicity of T6P as indicated for the corresponding deletion mutant in A. fumigatus. However, in A. niger as well as A. fumigatus and A. nidulans[12, 25], mutants of tppA are not totally lacking in trehalose. Therefore, it is possible that under specific conditions, e.g. when TppA is absent, TppB, and also TppC where present, may contribute to some T6P activity. Another possibility is that the sugar can be synthesized by proteins other than Tps/Tpp, e.g. the Trehalose Phosphorylase pathway, for which putative genes have been identified and partially characterized in N. crassa and A. fumigatus and also exist in A. niger (ANI_1_2720024). However, it is possible to generate mutants, within the homologous Tps/Tpp group, in A. fumigatus and A. nidulans that totally lack trehalose [11, 12]. Therefore, we believe that this is the only active trehalose synthesis pathway in Aspergilli. However, internal trehalose contents may not solely be dependent on the presence and expression of these six genes, as in S. cerevisiae there is a strong linkage between trehalose synthesis and the degrading trehalases  as well as evidences of posttranscriptional activation of the genes involved in trehalose metabolism [42, 43].
Besides a putative phosphatase activity, TppB and TppC may have similar biological roles as the yeast proteins Tps3 and Tsl1, which also contain phosphatase domains – in yeasts, deletion of both genes is necessary before some reduction in internal trehalose content can be observed . It is intriguing that tpsB and tppC are linked on the chromosome. We cannot explain why the conidial trehalose content in this double mutant was significantly higher after 28 days, but based on the expression patterns (see Figure 3), it is possible that the expression of the two genes are regulated by the same factors. In addition to the above-mentioned observations, some conclusions can be drawn from the gene expression data: All identified genes were expressed, indicating that the paralogs are not inactive duplicates. For tpsC and tppB, the expressions were consistently low after 6 h, indicating that the two genes may be regulated by the same mechanism. This assumption is supported by a previous observation using A. oryzae arrays where the tpsC and tppB orthologs were down-regulated in a deletion strain of atfA, a gene encoding a transcription factor . To our knowledge, two previous studies describing the expression of trehalose synthesis genes in A. niger during germination, using microarray technology, or in combination with RNA sequencing, have been published [29, 45]. With the exception that van Leeuwen and co-workers  saw a drastic drop after 2 h and then a gradual up-regulation of tpsA and tpsB, those results are in line with our findings.
The extensive measurements of internal trehalose indicate that the trehalose contents, for all strains, were low in 5 day old conidia, significantly elevated in 14 day old conidia, and then maintained at the value of 14 days (Figure 7). A plausible hypothesis is that conidia of A. niger reach full maturity, at least in terms of trehalose accumulation, sometime between 5 days and 2 weeks. Consequently it is not advisable to perform stress experiments on young conidia because their trehalose content is not necessarily typical for the final level, especially not in kusA deficient strains that seem to have slower conidial maturation in terms of trehalose content.
We found that 2 week old conidia of ΔtppB were more susceptible to heat shock than wild-type conidia, indicating that trehalose protects the spores from thermal stress. These results are in line with earlier studies in Aspergillus species [11, 12, 23]. However, in contrast to results from A. fumigatus and A. nidulans, we could not detect any increased sensitivity of ΔtppB to oxidative stress [11, 12], salt or acid stress, or any decreased viability after long term storage. It should be noted that unlike ΔtppB in our experiments, which harbored approximately one third of wild-type trehalose content, the A. fumigatus and A. nidulans mutants were totally depleted of trehalose.
In S. cerevisiae it has been shown that, using a two-hybrid assay, the four homologous proteins physically interact. When repeating the experiments using the six identified A. niger proteins, we could observe interactions for four of six proteins. These results suggest that TppA and TpsA-C form a complex, while the phylogenetically more distant proteins, TppB and TppC, are present outside the complex. However, due to the experimental limits, it is possible that neither TppB nor TppC was correctly folded and therefore not interacting. It is notable that in S. cerevisiae, a truncated version of Tsl1 was necessary for the success of the interaction experiments , in contrast to our experiment in which we only used full-length proteins.
To conclude, in this study novel information about the six gene products involved in trehalose synthesis in A. niger has been generated. When characterizing deletion mutants, lack of the most conserved trehalose phosphate synthase tpsA, the trehalose phosphate phosphatase tppA, or the previously non-characterized tppB, resulted in lower trehalose contents. An additional insight is that the components in a putative trehalose synthesis complex differ among the Aspergilli, but some gene products are common throughout the fungal kingdom.
Dr. Jonathan Hilmer for assistance with the T6P analysis and Dr. Su-lin Leong for proofreading the manuscript before submission, are greatly acknowledged. This work was financed by the Swedish research council Formas.
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