Effects of the addition of Mn2+ on growth and cellulase production of T. reesei
To determine how Mn2+ influences the hyphal growth, T. reesei Rut-C30 strains were cultured on MM (minimal medium) plates supplemented with different concentrations of Mn2+ (0, 1, 10, 20, and 40 mM final concentration) and 2% glucose as the sole carbon source. The mycelium length of T. reesei Rut-C30 after addition Mn2+ is shown in Fig. 1a. There was no significant difference in the hyphal growth with 1–10 mM Mn2+. However, when the concentrations of Mn2+ increased to 20 mM, the strains grew more slowly and sparsely. As shown in Fig. 1b, treatment with 20 mM Mn2+ caused a 30% reduction in the colony diameter compared with that of the untreated strains. Moreover, after treatment with 40 mM Mn2+, the treated strains showed a severe reduction in the colony diameter (53%).
To evaluate the effects of Mn2+ on cellulase production and total protein secretion, the same weight of precultured mycelia of T. reesei Rut-C30 was transferred to liquid MM containing 1% Avicel as the sole carbon source and different concentrations of Mn2+ (0, 1, 10, 20 and 40 mM). As shown in Fig. 1c (Additional file 8: Figure S7A), addition of Mn2+ at a final concentration of 10 mM significantly stimulated CMCase activity (representing endo-β-glucanase activity), with an increase of approximately 140% compared to the controls without addition of Mn2+. However, 1, 20, and 40 mM of Mn2+ did not evidently affect CMCase activity. As shown in Fig. 1d (Additional file 8: Figure S7B) and e, the addition of Mn2+ at a final concentration of 10–40 mM significantly stimulated pNPCase activity (representing exo-β-glucanase activity) and increased total protein concentration after 1 day of fermentation, with an increase of approximately 327 and 55%, respectively.
The above results demonstrated that 10–40 mM Mn2+ could stimulate cellulase production and total protein secretion in T. reesei Rut-C30, and that 20–40 mM Mn2+ could delay hyphal growth. The optimal concentration of Mn2+ to enhance cellulase production was 10 mM, which was selected for further research in our study. CMCase or pNPCase activity was directly used to represent cellulase activity in our study.
To further determine the effects of Mn2+ supplementation on the synthesis of cellulases or total protein secretion, the expression levels of four main cellulase genes (cbh1 encoding cellobiohydrolase I, cbh2 encoding cellobiohydrolase II, egl1 encoding endoglucanase I, and egl2 encoding endoglucanase II), and a transcriptional regulator of cellulases, xyr1, were compared by quantitative reverse-transcription PCR (RT-qPCR) after induction for 24, 48, and 72 h in cultures with 0 or 10 mM Mn2+ supplementation. The primers used to detect transcriptional changes of these genes are listed in Additional file 1: Table S1. The transcriptional levels of four main cellulase genes significantly increased by almost 2- to 3.5-fold, after 24, 48, and 72 h of induction following supplementation with 10 mM Mn2+ (see Additional file 2: Figure S1A–D). In T. reesei, XYR1 is a global transcriptional activator of cellulose and hemicellulase genes [27]. In accordance with the transcription of cbh1, cbh2 and egl1, egl2, the expression level of xyr1 was also significantly stimulated after 72 h of induction (see Additional file 2: Figure S1E). These results were consistent with the upregulation of cellulase activity through the addition of 10 mM Mn2+. However, when compared to expression of other cellulase-related genes, the delayed upregulation of xyr1 expression implied that other putative regulators may participate in Mn2+ metabolism/regulation to directly induce cellulase gene expression, besides indirect induction through xyr1.
Variation in intracellular and extracellular Mn2+ concentration after the addition of Mn2+
Extracellular Mn2+ can significantly augment the intracellular Mn2+ content, and subsequently affect the physiology and metabolism of G. lucidum [17]. To investigate whether enhanced cellulase production is linked to intracellular Mn2+ in T. reesei Rut-C30, the intracellular and extracellular Mn2+ concentrations were measured by inductively coupled plasma mass spectrometry (ICP-MS) during cultivation. As illustrated in Fig. 2a, the levels of intracellular and extracellular Mn2+ were almost constant in the control sample without Mn2+ addition. On the contrary, upon Mn2+ addition (10 mM final concentration), the intracellular Mn2+ concentration initially markedly increased, reaching its maximum at 24 h and then declined gradually, while the extracellular Mn2+ concentration dropped initially, reaching its minimum at 24 h and then increased gradually, suggesting that Mn2+ was transported into cells initially in response to a higher extracellular Mn2+ concentration. Subsequently, intracellular Mn2+ gradually effused into the medium from 24 h. We hypothesized that a mechanism could pump Mn2+ in and out of the cell.
Identification of Mn2+ transport proteins TPHO84-1 and TPHO84-2
Jensen et al. [16] suggested that PHO84, a low-affinity transporter of Mn2+ from Saccharomyces cerevisiae, transports Mn2+ when cells are exposed to higher Mn2+ concentrations. In our study, Mn2+ efficiently transported into T. reesei Rut-C30 cells at an Mn2+ concentration of 10 mM (Fig. 2a). To identify the protein(s) responsible for transporting Mn2+ in T. reesei, we conducted a homology search with the protein sequence of PHO84 (GenBank: KZV08715.1) in the T. reesei genome from the JGI database (http://genome.jgi.doe.gov/Trire2/Trire2.home.html). Five high score hits were obtained with the following protein identity matches: TRE77552, 59.6%, TRE81389, 51.8%, TRE45852, 49.0%, TRE45868, 40.1%, and TRE106118, 35.2%.
We hypothesized that Mn2+ transport proteins would be highly expressed during Mn2+ addition. To assess which proteins encoded putative Mn2+ transport activity, transcriptional levels of tre77552, tre81389, tre45852, tre45868 and tre106118 were monitored by RT-qPCR with 10 mM Mn2+ compared to no Mn2+ addition at different induction times. As illustrated in Additional file 3: Figure S2, expression of these genes was significantly upregulated in the samples treated with 10 mM Mn2+. Based on their induction at higher Mn2+ concentration, all five genes might be responsible for Mn2+ transport of T. reesei Rut-C30.
To further investigate whether any of these five proteins displayed a function similar to that of PHO84, involved in transport of Mn2+ in S. cerevisiae, the five deletion mutants, Δ77552, Δ81389, Δ45852, Δ45868, and Δ106118, respectively, were collected to measure intracellular Mn2+ concentrations by ICP-MS compared to that in the parental strain. As shown in Fig. 2b, Δ77552 and Δ45868 showed a marked decrease in steady-state levels of cellular Mn2+ when compared with the Rut-C30 strain, in which a 70–80% decrease in metal accumulation was obtained. However, under the same conditions, Δ81389, Δ45852, or Δ106118 had an Mn2+ concentration similar to that in Rut-C30 strain (see Additional file 4: Figure S3). The tre77552 and tre45868 double mutant strain Δtpho84-1/2 showed lower intracellular Mn2+ concentration than each single mutant (Fig. 2b). Δ77552 and Δ45868 were also complemented by transforming vectors ptpho84-1-rc and ptpho84-2-rc into them, respectively (see Additional file 5: Figure S4). Complementation strains (tpho84-1-rc and tpho84-2-rc) were obtained to demonstrate the restoration of Mn2+ transport with intracellular Mn2+ concentrations similar to those of the parent strain Rut-C30 (Fig. 2b). The results demonstrated that the intracellular Mn2+ concentration can be increased via putative Mn2+ transport proteins TRE77552 and TRE45868 in T. reesei Rut-C30.
As predicted by SMART (http://smart.embl-heidelberg.de/), both TRE77552 and TRE45868 are membrane proteins with an 11- and 9-transmembrane domain topology, respectively. To visualize the location of TRE77552 and TRE45868, we constructed two chimeric proteins, RFP-77552 and RFP-45868, by fusing red fluorescence protein to their N-terminus (see Additional file 6: Figure S5A). The two chimeric proteins were overexpressed via cbh1 promoter, which allowed us to confirm that TRE77552 and TRE45868 are located at the mycelial surface. Additional file 6: Figure S5B shows the in vivo epifluorescence analysis of the two chimeric protein (RFP-77552 and RFP-45868) transformants, rfp-tpho84-1 and rfp-tpho84-2, respectively, depicting strong and stable fluorescent signal at the mycelial surface, as expected for plasma membrane proteins. However, overexpression of genes encoding membrane bound proteins by a strong promoter might cause some uncertainty.
Based on these findings, we considered that both tre77552 and tre45868 encode Mn2+ transports with Mn2+ transport function located at the plasma membrane, and named the two genes as tpho84-1 and tpho84-2, respectively. Intracellular Mn2+ can be transported into T. reesei Rut-C30 cells via TPHO84-1 and TPHO84-2. It is presently unclear whether tre81389, tre45852, and tre106118 are inactive in the transport of Mn2+.
Role of TPHO84-1 and TPHO84-2 in cellulase production
Δ77552 (Δtpho84-1) and Δ45868 (Δtpho84-2), and their complementation strains tpho84-1-rc and tpho84-2-rc were used to determine the effect of TPHO84-1 and TPHO84-2 in mediating the growth and cellulase production in T. reesei Rut-C30. To determine the effect of TPHO84-1 and TPHO84-2 on mediating growth, Rut-C30, Δtpho84-1, Δtpho84-2, tpho84-1-rc, tpho84-2-rc and Δtpho84-1/2 strains were cultured on minimal medium plates adding 0 or 10 mM Mn2+ and 2% glucose as the sole carbon source. The mycelium morphology is shown in Fig. 3a. There was no significant effect on the hyphal growth of Rut-C30, Δtpho84-1, Δtpho84-2, tpho84-1-rc and tpho84-2-rc strains at 0 or 10 mM Mn2+. However, the hyphal growth of the Δtpho84-1/2 transformant was slightly repressed, compared with that of the parent strain at both 0 and 10 mM Mn2+ (Fig. 3b).
To further test the effect of Mn2+ transport proteins on cellulase production, we investigated the effect of Rut-C30, Δtpho84-1, Δtpho84-2, tpho84-1-rc, tpho84-2-rc, and Δtpho84-1/2 strains on cellulase production after supplementation of 0 or 10 mM Mn2+. There was no obvious difference in CMCase (see Additional file 7: Figure S6A) and pNPCase (see Additional file 7: Figure S6B) activities between these six strains without Mn2+ addition. However, Δtpho84-1 and Δtpho84-2 strains showed approximately 30–40% reduction in CMCase activity, compared with the parental strain Rut-C30 with 10 mM Mn2+ addition. Furthermore, Δtpho84-1/2 strain had lower CMCase activity than each single mutant, at approximately 50% reduction compared with Rut-C30 (Fig. 3c and Additional file 8: Figure S8A). Similarly, Δtpho84-1 and Δtpho84-2 strains showed approximately 25 to 30% reduction in the pNPCase activity, compared with the parental strain Rut-C30, with 10 mM Mn2+ addition. Furthermore, the Δtpho84-1/2 strain showed lower pNPCase activity than each single mutant, and approximately 40% reduction compared with Rut-C30 (Fig. 3d and Additional file 8: Figure S8B). The cellulase production capabilities of tpho84-1-rc and tpho84-2-rc were complemented at 10 mM Mn2+ supplementation compared to parental strains Δtpho84-1 and Δtpho84-2, respectively, to a level similar to that of the original strain Rut-C30 (Fig. 3c, d and Additional file 8: Figure S8A, B).
Additionally, RT-qPCR was performed to determine the transcription levels of the important cellulase genes cbh1 and egl1 in the T. reesei Rut-C30, Δtpho84-1, Δtpho84-2, tpho84-1-rc, tpho84-2-rc, and Δtpho84-1/2 mutants. The primers used to detect transcriptional levels of these genes are listed in Additional file 1: Table S1. In agreement with the CMCase activity levels, no obvious difference in transcription levels of cbh1 and egl1 between above six strains was observed without Mn2+ addition (data not shown). However, the deletion strains Δtpho84-1, Δtpho84-2, and Δtpho84-1/2 showed a marked reduction in cellulase transcription compared with the parental strain Rut-C30 upon 10 mM Mn2+ addition at all time points examined (Fig. 3e, f). Similarly, the expression levels of the cbh1 and egl1 genes of tpho84-1-rc and tpho84-2-rc were complemented under Mn2+ supplementation, to a level similar to that of the original strain Rut-C30.
These results indicate that TPHO84-1 and TPHO84-2 participate in inducing cellulase production of T. reesei Rut-C30 only under Mn2+ addition, which is in accordance with its function as Mn2+ transport protein.
Increase in cytosolic Ca2+ level and calcium signaling after Mn2+ addition
Next, we investigated how Mn2+ addition can upregulate cellulase gene expression via Mn2+ transport proteins TPHO84-1 and TPHO84-2. Using Fluo-3/AM fluorescent dye, a dye that only emits green fluorescence after crossing the cell membrane and binding with Ca2+ [28], whose intensity represents relative amounts of free intracellular Ca2+ [29], we found that the cytosolic Ca2+ concentration was increased after Mn2+ addition. As shown in Fig. 4A, a stronger green fluorescence intensity was observed in the Rut-C30 cells under 10 mM Mn2+ supplement on the second day than that observed with the control (no Mn2+ supplement), demonstrating that Mn2+ leads to an increase in the level of cytosolic Ca2+. The fluorescence level emitted by the Ca2+-activated fluorochrome reached a 2.23-fold increase under 10 mM Mn2+ addition compared to that in the control (Fig. 4B). These results indicate that Mn2+ induced an increase in the concentration level of cytosolic Ca2+. A similar phenomenon was reported in S. cerevisiae [30].
We measured the level of cytosolic Ca2+ in the Δtpho84-1, Δtpho84-2, and Δtpho84-1/2 strains with Mn2+ addition. As illustrated in Fig. 4A, B, the significant increase in intracellular Ca2+ induced by Mn2+, observed in parental strain Rut-C30, was absent in the Δtpho84-1, Δtpho84-2, and Δtpho84-1/2 strains. The augmentation of cytosolic Ca2+ levels induced by Mn2+ was blocked in the Δtpho84-1, Δtpho84-2, and Δtpho84-1/2 strains. These results suggest that rising cytosolic Ca2+ levels depend on Mn2+ transport into Rut-C30 cells via TPHO84-1 and TPHO84-2. This also indicates that Ca2+ influx in cells is closely associated with Mn2+ homeostasis.
Previous studies have demonstrated that the calcium signal transduction pathway can upregulate cellulase gene expression [13]. We investigated whether the increased levels of cytosolic Ca2+, induced by Mn2+, can trigger calcium signal transduction pathways in T. reesei. To test our hypothesis, RT-qPCR was carried out to analyze the transcriptional levels of calcium signaling-related genes, including calmodulin (cam, GenBank: ACZ26150.1), calcineurin (cna1, GenBank: EGR49476.1) [31], and calcineurin-responsive zinc finger transcription factor 1, crz1 [13], under Mn2+ addition. The primers used to detect transcription of these genes are listed in Additional file 1: Table S1. As shown in Fig. 4C–E, similar to the increase in the contents of cytosolic Ca2+, expression of these genes was significantly upregulated with Mn2+ addition. These results suggested that Mn2+ can increase the concentration of cytosolic Ca2+, thus stimulating the calcium signal transduction pathway to induce cellulase production in T. reesei Rut-C30.
Mn2+ induces cellulase production via cytosolic Ca2+
To investigate the assumption that Mn2+ induces cytosolic Ca2+ improvement and cellulase production via Ca2+ channels, we used LaCl3, a plasma membrane Ca2+ channel blocker to prevent influx of external Ca2+ [32]. Figure 5A, B shows that the Fluo-3/AM fluorescence intensity of mycelia remarkably reduced almost 60% with LaCl3 compared with no LaCl3 addition, under 10 mM Mn2+. The increased content of cytosolic Ca2+ induced by Mn2+ could be effectively attenuated by adding LaCl3 to T. reesei Rut-C30. Meanwhile, the increased expression of calcium signaling-related genes, cam, cna1, and crz1, which are induced by 10 mM Mn2+, was also effectively prevented by adding LaCl3 (data not shown).
Ca2+ participates in regulating cellulase production in T. reesei [13]. To investigate whether a cellulase increase, induced by Mn2+, was blocked by LaCl3, we analyzed CMCase and pNPCase activities and transcription of key cellulase genes cbh1 and egl1. As shown in Fig. 5C, D (Additional file 8: Figure S9A, B), under Mn2+ supplementation and with LaCl3, the CMCase and pNPCase activities in T. reesei Rut-C30 decreased by 50 and 49%, respectively, compared with no LaCl3 addition. Meanwhile, the expression levels of cbh1 and egl1 were reduced by 60% at 72 h (Fig. 5E, F). However, there was no obvious change to CMCase activities and transcriptional levels of cbh1 and egl1 with or without LaCl3 when Mn2+ was not added (Fig. 5C–F, Additional file 8: Figure S9A, B). These results showed that LaCl3 could significantly decrease the Mn2+-induced high expression levels of key cellulase genes in T. reesei Rut-C30.
These data indicated that Mn2+ induced cytosolic Ca2+ increase via the Ca2+ channel. When a Ca2+ channel blocker LaCl3 was added, the increase in cytosolic Ca2+ concentration and cellulase production induced by Mn2+ were effectively attenuated.
To investigate whether a cellulase increase, induced by Mn2+, is associated with calcium signal transduction, we constructed a crz1 deletion mutant Δcrz1 as Chen et al. [13] to block the calcium signal transduction pathway. As shown in Fig. 6a, b (Additional file 8: Figure S10A, B), the remarkable increase of CMCase and pNPCase activities induced by Mn2+, observed in parental strain Rut-C30, was effectively attenuated by deleting crz1. Similarly, the transcriptional levels of cbh1 and egl1 were markedly reduced in the Δcrz1 mutant at all time points examined (Fig. 6c, d).
Taken together, these data indicated that Mn2+ induces cellulase improvement via calcium signal transduction in T. reesei Rut-C30. The improvement of cellulase production induced by Mn2+ is effectively prevented in a crz1 mutant, blocking the calcium signal transduction pathway.
TPMR1 functions in conjunction with channels of Mn2+ and Ca2+
Mn2+/Ca2+ homeostasis exists in other fungi [33,34,35,36]. PMR1 plays an important role in Mn2+/Ca2+ homeostasis, and may act as a Ca2+/Mn2+ exchanger to balance Mn2+ via Ca2+ [17]. To clarify how Mn2+ increased cytosolic Ca2+ concentration, using Neurospora crassa PMR1 (GenBank: CAB65296.1) as the query, we searched for a PMR1 homolog (TRE119592) in the T. reesei genome, named as TPMR1.
We constructed a tpmr1 deletion mutant Δtpmr1. To investigate whether TPMR1 is responsible for pumping in Ca2+ upon 10 mM Mn2+ addition in T. reesei, we measured the level of intracellular Ca2+ in the Δtpmr1 strain with or without Mn2+ addition. As shown in Fig. 7A and B, the increase in intracellular Ca2+ induced by Mn2+ in Rut-C30 was absent in the Δtpmr1 strain. The augmentation of Ca2+ levels induced by Mn2+ was blocked in the Δtpmr1 strain. These results suggested that TPMR1 is responsible for the increase of cytosolic Ca2+ under Mn2+ addition in T. reesei Rut-C30.
To further investigate the function of TPMR1 in conjunction with Mn2+ and Ca2+ channels, the intracellular and extracellular Mn2+ concentrations in the Δtpmr1 strain with or without Mn2+ addition were measured by ICP-MS during cultivation. As illustrated in Additional file 9: Figure S12, upon Mn2+ addition (10 mM final concentration), the intracellular Mn2+ concentration initially markedly increased, reaching and maintaining a high level after 24 h. The extracellular Mn2+ concentration dropped initially, reaching its minimum from 24 to 72 h. The intracellular and extracellular Mn2+ concentrations of the Δtpmr1 strain (Additional file 9: Figure S12) are quite different from that of Rut-C30 (Fig. 2a), whose intracellular Mn2+ gradually effused into the medium. These results suggested that TPMR1 is responsible for pumping Mn2+ out of the cell.
To further test the effect of TPMR1 on cellulase production under Mn2+ addition, we compared the cellulase production in parental strain Rut-C30 and Δtpmr1 mutant. Upon the addition of Mn2+, the Δtpmr1 strain caused about 26 and 51% reductions in the CMCase and pNPCase activities, respectively, compared with that of Rut-C30 (Fig. 7C, D and Additional file 8: Figure S11A, B). Moreover, the transcriptional levels of cbh1 and egl1 observed are in agreement with the CMCase and pNPCase activity levels, which were markedly reduced in the Δtpmr1 mutant at all time points examined (Fig. 7E, F). The increase in cellulase production induced by Mn2+ was remarkably weakened in the tpmr1 deletion mutant. These results demonstrated that TPMR1 functions in conjunction with channels of Mn2+ and Ca2+ in T. reesei Rut-C30.