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protein Pga1 regulates biosynthesis of penicillin, chrysogenin and roquefortine in Penicillium chrysogenum
1 Instituto de Biotecnología de León, INBIOTEC, Parque Científico de León, León 24006, Spain
2 Área de Microbiología, Fac. CC. Biológicas y Ambientales, Universidad de León, León 24071, Spain
Correspondence
Juan F. Martín
jf.martin{at}unileon.es
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ABSTRACT |
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Present address: Departamento de Microbiología, Facultad de Ciencias Básicas, Universidad de Pamplona, Campus Universitario, Pamplona, Colombia.
Present address: División de Ciencias Biológicas y de la Salud, Universidad Autónoma Metropolitana, UAM-Xochimilco, México D.F. 04960, Mexico.
Two supplementary tables and two supplementary figures are available with the online version of this paper.
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INTRODUCTION |
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) and secondary metabolism (Calvo et al., 2002).
G proteins consist of three subunits, 
, β and 
; they remain inactive in the heterotrimeric state with GDP bound to the subunit, but become activated by a guanine nucleotide exchange in response to G-protein-coupled receptor activation. When GTP is bound to the subunit, as a result of the nucleotide exchange, this subunit dissociates from the β dimer, and the subunit and β dimer then interact with downstream effectors in the signal cascade (Hamm, 1998). Certain amino acid changes in the switch region of G subunits produce mutant subunits unable to separate from the β dimer, resulting in constitutively inactivated G proteins (Bohm et al., 1997), as occurs in the subunit FadAG203R of Aspergillus nidulans (Kurjan, 1992). On the other hand, mutations affecting the endogenous GTP hydrolase (GTPase) activity result in dominant activating subunits that become permanently bound to GTP, for instance the A. nidulans mutant subunit FadAG42R (Yu et al., 1996).
In fungi, G proteins have been implicated in mediating several processes, including differentiation and virulence (reviewed by Lengeler et al., 2000; Li et al., 2007).
Particularly interesting is the role of G proteins in transduction of environmental signals that control secondary metabolite biosynthesis (Tag et al., 2000; Calvo et al., 2002). Filamentous fungi are well-known producers of antibiotics, mycotoxins, pigments and other secondary metabolites (Martín, 2000; Keller et al., 2005). Sequencing of the genome of several fungi, including Aspergillus niger (Galagan et al., 2005), Aspergillus fumigatus (Nierman et al., 2005), Aspergillus oryzae (Machida et al., 2005) and Penicillium chrysogenum (M. A. van den Berg and others, unpublished) has revealed an impressive wealth of secondary metabolite gene clusters (Zhang et al., 2004; Hoffmeister & Keller, 2007).
P. chrysogenum is an important filamentous fungus because of its ability to produce large amounts of penicillin (Martín, 1998; Elander, 2003). The biochemistry and molecular genetics of penicillin biosynthesis have been widely studied (reviewed by Aharonowitz et al., 1992; Martín, 2000; Fierro et al., 2002; Liras & Martín, 2006) and the DNA region encoding the penicillin biosynthesis genes has been fully sequenced (Fierro et al., 2006; van den Berg et al., 2007). The regulation of expression of the penicillin biosynthesis genes has been studied (reviewed by Brakhage, 1998; Martín, 2000), but the signal transduction cascade that connects environmental factors to penicillin gene expression remains unknown.
In addition to penicillin, P. chrysogenum synthesizes the yellow pigment chrysogenin and the mycotoxin roquefortine. Chrysogenin is produced by the wild-type strain P. chrysogenum NRRL 1951 (Asilonu et al., 2000), but its synthesis has been drastically reduced by mutations in the Wis 54-1255 mutant and in all industrial strains, because it is an undesired product that hinders penicillin purification. Although the biosynthetic pathway of this polyketide-derived product is unknown, its easy spectrophotometric detection makes it a valuable marker to study regulation of secondary metabolite production.
Roquefortine is a dimethylallyltryptophan-derived metabolite known to be produced in Penicillium roqueforti and several other fungi (Rundberget et al., 2004; Frisvad & Filtenborg, 1983; de la Campa et al., 2007), and we have identified this mycotoxin in cultures of different P. chrysogenum strains. The gene cluster responsible for the biosynthesis of this mycotoxin is still being elucidated (A. Gómez & J. F. Martín, unpublished results). The roquefortine HPLC assay allows a reliable quantification of this mycotoxin that constitutes another interesting model for studying G-protein-mediated regulation of secondary metabolites in this fungus.
We have previously cloned the pga1 gene of P. chrysogenum encoding a Gi subunit of a heterotrimeric G protein (García-Rico et al., 2007) and have studied the effect of dominant activating (pga1G42R) and dominant inactivating (pga1G203R) mutations on growth and differentiation, finding that Pga1 negatively regulates conidation of P. chrysogenum mainly by a cAMP-independent mechanism (García-Rico et al., 2008). It was, therefore, of great interest to study the effect of these opposite mutations of the pga1 gene on the biosynthesis of different P. chrysogenum secondary metabolites (a β-lactam, a polyketide and a dimethylallyltryptophan derivative), to gain insight into the signal transduction cascade that controls expression of the three different types of secondary metabolites in this fungus.
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METHODS |
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subunit in the production of the secondary metabolites; these strains were obtained by genetic manipulation of P. chrysogenum NRRL 1951 and Wis54-1255, and are summarized in Table 1.
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). Strains PgaG42R-1, PgaG42R-3 and PgaG42R-4 were obtained by transformation of P. chysogenum Wis54-1255 with plasmid pPgaG42R (García-Rico et al., 2007), and strains GpdG42R-1, GpdG42R-3 and GpdG42R-5 were obtained by transformation of P. chysogenum Wis54-1255 with plasmid pGpdG42R, containing the dominant activating allele pga1G42R expressed from the A. nidulans gpdA promoter (García-Rico et al., 2007).
Transformants of P. chrysogenum NRRL 1951 and P. chysogenum Wis54-1255 with plasmid pJL43b1 (García-Rico et al., 2007), named NRRL-control and Wis-control respectively, were used as controls in the secondary metabolite production experiments; pJL43b1 was used as vector for constructions with the different pga1 alleles but contains no pga1. Fungal transformation was performed as described previously (García-Rico et al., 2007)
Culture conditions for penicillin production.
Cultures in flasks were performed as follows. Conidia of P. chrysogenum were collected from plates of Power medium (Fierro et al., 1996) and inoculated at a concentration of 1x107 conidia ml–1 in flasks with 50 ml defined inoculum medium (Casqueiro et al., 1999), which were incubated in an orbital shaker at 250 r.p.m., 25 °C for 36 h. Then 8 ml of this seed culture was transferred, in duplicate, to flasks with 100 ml lactose-containing (3 %, w/v) complex production (CP) medium (Kosalkova et al., 2000), and incubated at 250 r.p.m., 25 °C for 144 h. Every 24 h, samples of 5 ml were taken to determine penicillin G, pH and dry weight.
Fermentation in bioreactors at controlled pH was carried out as follows. Seed cultures were developed as indicated above in a total volume of 290 ml. The whole seed culture volume was inoculated into 3100 ml CP medium in 5 l fermenters (Biostat B, Braun), which were maintained at a constant pH of 6.8, 25 °C and stirrer speed of 350 r.p.m. for 144 h. Every 24 h, samples of 10 ml, in duplicate, were taken to determine penicillin G and dry weight, as described previously (García-Rico et al., 2007).
Extraction and quantification of chrysogenin.
Chrysogenin was extracted from solid cultures in CYA medium (7 days old) of the different strains of P. chrysogenum. The agar cultures of two plates of each strain were collected in 50 ml tubes and extracted with ethyl acetate containing 0.5 % formic acid (Nielsen & Smedsgaard, 2003).
The extraction was performed for 30 min with an ultrasonic treatment (50/60 Hz, P Selecta Ultrasons) to disrupt the agar pieces. After the extraction the organic extract was collected by centrifugation at 4000 r.p.m. for 10 min at 4 °C. The extraction procedure was repeated twice and the mixture of both extracts was dried in a vacuum evaporator (Rota-Vapour 210, Buchi). The dry solid residue was redissolved in 1 ml methanol, centrifuged and used for spectrophotometric determination at 280 nm and 400 nm, corresponding to the UV and visible light absorbance peaks of chrysogenin (Asilonu et al., 2000).
Quantification of roquefortine.
Roqueforine C in liquid cultures of the different P. chrysogenum strains in YES medium (Scott et al., 1970) was quantified by HPLC. Samples (10 ml) were taken from duplicate flasks at 24, 48, 72, 96 and 144 h of incubation, and centrifuged at 4500 g for 10 min to separate the soluble roquefortine in the supernatant from the mycelium-associated mycotoxin. The mycelium was washed twice with 0.9 % NaCl, placed into Falcon tubes and extracted with 20 ml dichloromethane for 30 min with ultrasound treatment to disrupt the mycelial pellets.
The extract was evaporated under vacuum in a rotary concentrator (Rota-Vapour, Buchi). The dry residue was redissolved in 120 µl methanol, centrifuged and used for HPLC analyses.
Chromatographic determinations of roquefortine in the mycelial extracts or in the aqueous culture supernatant were performed in a Shimadzu HPLC system consisting of an SPD-M10Avp photodiode array detector, a solvent delivery module, a system controller and an autoinjector. All analyses were carried out in a Symmetry (Waters) reverse-phase C18 column (150 mmx3.9 mm) of particle size 5 µm. Elution of roquefortine was performed at room temperature with a mobile phase of acetonitrile/water containing 0.04 % trifluoroacetic acid (flow rate 0.7 ml min–1). A linear gradient, from 70 : 30 to 20 : 80 (v/v) water/acetonitrile in 25 min, was used for separation. After 5 min elution with the 20 : 80 mixture the eluent composition was changed to the starting conditions. A standard curve of pure roquefortine (provided by Institute Biomar, León, Spain) was made as control for the quantitative determinations.
Induction of high intracellular cAMP concentrations.
High intracellular cAMP concentrations were induced in strain P. chrysogenum Wis54-1255 by addition of theophylline (Sigma-Aldrich) as described previously (García-Rico et al., 2008). Intracellular cAMP concentrations were determined as described previously (García-Rico et al., 2008).
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RESULTS |
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). As controls, the wild-type strain NRRL 1951 and a transformant with the plasmid vector without pga1 alleles (NRRL-control) were used.
All transformants expressing the constitutively activated Pga1G42R subunit produced clearly higher levels of penicillin (200–260 % with respect to the control); the increased production was observed from 72 h to 120 h of fermentation (Fig. 1a). Transformants with the constitutively inactivated Pga1G203R subunit showed production levels similar to those of the wild-type strain, and the same result was observed in strain AS-1 expressing a pga1 antisense RNA. These two strains cause an increase in the pH of the medium (Fig. 1a lower-right panel), an effect also observed in pga1-inactivated derivatives of strain Wis54-1255 (Fig. 2c), whose implications will be analysed in the following sections.
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) (see also Supplementary Table S1, available with the online version of this paper). This increased expression was already observed in cultures after 48 h of cultivation, and was higher (about fourfold in both genes) at 72 h, coinciding with the phase of high penicillin production. In contrast, the transformant expressing the constitutively inactivated Pga1G203R subunit showed no significant differences in the expression levels of either of the genes at any time, as compared to the wild-type NRRL 1951.
Stimulatory effect of the pga1G42R allele expressed from different promoters on penicillin biosynthesis by the improved penicillin producer strain Wis54-1255
P. chrysogenum Wis54-1255 produces significant higher levels of penicillin than the wild-type strain NRRL1951, although it still contains a single copy of the penicillin gene cluster (Fierro et al., 1995). Studies on the effect of the mutations of the G subunit on penicillin biosynthesis were initially performed in shake flasks and later in fermenters under pH-controlled conditions, to avoid the effect of pH changes on penicillin gene expression (Gutiérrez et al., 1999; Suárez & Peñalva, 1996).
Two different constructions to express the pga1G42R allele were used in this study: transformants PgaG42R, which contain the pga1G42R allele under the control of its own promoter, and transformants GpdG42R, in which the pga1G42R allele was expressed from the strong constitutive promoter of the gpdA (glyceraldehyde-3-phosphate dehydrogenase) gene. Three transformants with each of these promoters were studied in comparison with the parental strain Wis54-1255 and a control transformant containing the vector without pga1 insert (Wis-control).
Results from shaking flask cultures showed that the six transformants containing the pga1G42R allele produced increased levels of penicillin at 72–120 h of incubation, 50–60 % higher than the untransformed Wis 54-1255 strain (Fig. 2a, b). The stimulatory effect was similar in both constructions with the gpdA or pga1 promoters, although the accumulation of penicillin was faster at early times (24–48 h) in the constructions with the gpdA promoter (Fig. 2b).
The lower relative increase in penicillin production in the P. chrysogenum Wis54-1255-derived transformants as compared with transformants of the wild-type NRRL 1951 (200–260 %; Fig. 1a) suggests that the increase in production in the improved Wis54-1255 strain may be due to mutations introduced during the strain improvement programme that result in a positive effect in the activity of the G-protein-controlled cascade; therefore, the influence of exogenous copies of pga1G42R is lower in the higher penicillin producer Wis 54-1255.
Transcriptional studies of the pcbC and penDE genes in the Wis54-1255 transformants showed that there is a clear increase in the expression of both pcbC and penDE in the transformants carrying the pga1G42R allele expressed from either the pga1 or the gpdA promoters (transformant PgaG42R-1 is shown in Fig. 3, lanes 2), ranging from about 1.6-fold at 24 h to about 2.2-fold at 72 h of culture in both genes, as observed by densitometric analysis of the films (Supplementary Table S2). These results support the conclusions on the positive effect of constitutively activated G protein on penicillin gene expression obtained with the wild-type strain NRRL 1951.
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pga1 on the other side (pH values above 7.0) during the first 72 h of fermentation in shake flask cultures (Fig. 2c, lower panel).
To exclude a possible effect of pH changes on penicillin gene expression and production (Gutiérrez et al., 1999; Suárez & Peñalva, 1996), experiments were performed in four identical 5 l Braun Biostat B fermenters under constant pH values (pH adjusted to 6.8).
The kinetics of penicillin biosynthesis in the fermenters showed a consistent increase in penicillin production by the PgaG42R-1 transformant as compared to the parental Wis54-1255 strain (Fig. 4a); the increase was evident after 48 h of cultivation and reached about 50 % at 96 and 120 h.
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subunit, and the pga1 strain showed a decrease of penicillin production as compared to the parental Wis54-1255 strain; the two strains produced about 65 % (48 h) and 85 % (96 h) of the amount of penicillin in the control strain Wis54-1255.
This result demonstrates that the Pga1 subunit positively regulates penicillin production in P. chrysogenum, and also shows the influence of pH on the production. The fact that strains G203R-T and pga1 produce similar amounts of the antibiotic to the parental Wis54-1255 strain in shake flask cultures (Fig. 2c, upper panel) is due to the increase in the pH of the medium caused by these mutations. When pH values are kept at 6.8, the penicillin production falls to 65–85 % of the amount produced by Wis54-1255, and then the negative effect of inactivating the Pga1 subunit becomes evident.
Pga1 controls penicillin production by regulating expression of the penicillin biosynthetic genes
Expression of the pcbC and penDE genes in the pH-controlled fermenters was clearly enhanced in the PgaG42R-1 strain (Fig. 4b, lanes 3) as compared to the parental Wis54-1255 strain throughout the fermentation, especially in the first 24–48 h (4-fold and 3.5-fold respectively at 24 h) (Table 2). Expression of the first gene (pcbAB) of the penicillin pathway was also clearly stimulated in the PgaG42R-1 strain (Supplementary Fig. S1), but the degradation of the large (11.5 kb) pcbAB transcript prevented its quantification.
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, lanes 1) and strain pga1 (Fig. 4b, lanes 4), which was confirmed by densitometric analysis of the Northern hybridization (Table 2). This decreased expression correlated with the reduction in penicillin production observed in these strains (Fig. 4a). These results show that the Pga1 subunit controls penicillin production by regulating expression of the penicillin biosynthetic genes.
It is interesting to note that the decrease in the expression of the penicillin genes observed in strain pga1 does not take place in shake flask cultures (Fig. 3, lanes 3), which is explained by the activation of penicillin gene expression caused by the increase in the pH values in this strain (Gutiérrez et al., 1999; Suárez & Peñalva, 1996).
Increase of intracellular cAMP levels has no effect on penicillin production
We have previously reported that cAMP is a secondary messenger in the Pga1-mediated signalling pathway (García-Rico et al., 2008). Pga1 positively regulates cAMP intracellular levels, and the conidiation process is partially regulated via cAMP. To test wether cAMP has a role in the regulation of penicillin production by Pga1, we used the phosphodiesterase inhibitor theophylline to increase intracellular cAMP concentrations.
Shake flask fermentations of strain Wis54-1255 were carried out in 10 mM theophylline-supplemented and non-supplemented CP medium. The presence of 10 mM theophylline caused an increase in the intracellular cAMP concentration of 52–64 % (Table 3). However, this increase in the cAMP concentration had no effect on penicillin production throughout the fermentation (Supplementary Fig. S2). This result suggests that the regulation of penicillin biosynthesis by Pga1 may not be mediated by cAMP.
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), particularly in solid cultures in CYA and Power media. The production of this pigment was higher in the strains containing the pga1G42R allele as compared to the wild-type NRRL 1951 (Fig. 5b). These results were confirmed by extracting the pigment from solid cultures in CYA medium. The transformant expressing the constitutively activated Pga1G42R subunit showed a twofold increase in chrysogenin content as compared to the wild-type strain. Transformants with the constitutively inactivated Pga1G203R subunit produced an amount of chrysogenin slightly higher than that of the wild-type strain (Fig. 5c). Therefore, the biosynthesis of chrysogenin is stimulated by a constitutively activated G protein, as occurs with penicillin.
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The pga1G42R mutation stimulates roquefortine production in P. chrysogenum
P. chrysogenum Wis 54-1255 produced low levels of roquefortine in liquid cultures in YES medium (optimal for roquefortine biosynthesis) as compared to P. roqueforti (A. Gómez & J. F. Martín, unpublished results). The roquefortine produced by P. chrysogenum Wis 54-1255 remained mycelium-associated for 72 h and then was released into the culture medium at 96 h, coinciding with a change in pH and other fermentation parameters. The mycelium-associated form does not appear to be intracellular, but remains adhered to the fungal cell wall.
When the production of both mycelium-associated and extracellular roquefortine was quantified in the strains containing the different pga1 mutations (Fig. 6), it was found that the transformant expressing the pga1G42R allele produced consistently higher levels of roquefortine throughout the fermentation as compared to the parental strain Wis 54-1255, except at day 3, where the sum of mycelium-associated and released roquefortine showed no significant differences between the two strains. The mycelium-associated roquefortine reached a peak at 72 h and then at 96 h was released into the supernatant, achieving levels of 440 µg extracellular roquefortine per g dry weight in the P. chrysogenum pga1G42R transformant as compared to 350 µg per g dry weight for the parental strain. The transformant expressing the dominant inactivating pga1G203R allele and the deletion mutant pga1 accumulated less mycelium-associated roquefortine at 72 h than the parental strain; and the released roquefortine at 96 h and thereafter was also lower in these two strains. These results indicate that expression of the constitutively activated Pga1G42R subunit increases production and secretion of roquefortine in P. chrysogenum, whereas formation of the constitutively inactivated Pga1G203R subunit or deletion of the pga1 gene leads to lower accumulation of this mycotoxin. The stimulating effect of Pga1G42R on roquefortine production was lower than the effect exerted by this mutant allele on the biosynthesis of penicillin and chrysogenin.
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DISCUSSION |
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subunits homologous to Pga1 of P. chrysogenum have been described in several filamentous fungi (García-Rico et al., 2007), but the effect of alteration of G subunits on the biosynthesis of secondary metabolites has been described only in a few cases (Hicks et al., 1997; Calvo et al., 2002; Yu & Keller, 2005). The results known so far indicate that G subunits differentially regulate secondary metabolite biosynthesis; in some cases it appears to be a positive and in others a negative regulation. In Neurospora crassa the expression of a constitutive active Gna-1 G subunit caused a reduced secretion of carotenoid pigments (Yang & Borkovich, 1999), in contrast to Penicillium marneffei, where transformants expressing the dominant activating gasAG42R allele showed an increased production of the red pigment characteristic of this species (Zuber et al., 2002). Even within the same species there are examples of differential regulation of secondary metabolites by G subunits, as happens in A. nidulans (Tag et al., 2000) and in Trichoderma atroviride, in which the G subunit Tga1 has opposite roles in regulation of the biosynthesis of different antifungal substances (Reithner et al., 2005).
Some fungi produce a variety of secondary metabolites, including antibiotics, mycotoxins and pigments (Zhang et al., 2004; Hoffmeister & Keller, 2007), and in most cases it is unknown if all secondary metabolites respond in the same way (positively or negatively) to regulation by G subunits.
As shown in this article, dominant activating mutations (G42R) of the P. chrysogenum pga1 gene led to increased production of penicillin, roquefortine and the yellow pigment chrysogenin. On the other hand, the dominant inactivating mutation pga1G203R and the deletion of the pga1 gene resulted in a reduction of penicillin production and a decrease of roquefortine accumulation after the third day of fermentation, but these mutations did not abolish production of the secondary metabolites and expression of their genes. Therefore, although the dominant activating Pga1 G subunit increases the expression of secondary metabolism genes, Pga1 is not strictly required for secondary metabolite production. Since the dominant inactivating G203R mutation does not cause a reduction of chrysogenin levels, this pigment seems to be less sensitive to Pga1 regulation. Alternatively, cycling of Pga1 between GTP- and GDP-bound forms may be required to regulate chrysogenin accumulation properly. A model of the network of regulatory effects of Pga1 on coniditation (García-Rico et al., 2008) and secondary metabolite biosynthesis is depicted in Fig. 7.
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). Later studies showed that FadA has a positive effect on penicillin production in A. nidulans, i.e. the effect of the dominant activating fadAG42R allele is opposite on aflatoxin production and penicillin biosynthesis in this fungus (Tag et al., 2000). The same mutant allele in Aspergillus flavus caused a decrease in the production of aflatoxin and cyclopiazonic acid (Calvo et al., 2002), but an increase of trichothecene production in Fusarium sporotrichioides (Tag et al., 2000).
The reason for the differential positive or negative effect of the dominant activating G subunit on different secondary metabolities is largely unknown and requires detailed transcriptional analysis and characterization of the different G subunit targets. As reported in this article, in P. chrysogenum the dominant activating G subunit increases the expression of the three penicillin biosynthetic genes pcbAB, pcbC and penDE. It is unknown how the transcription-enhancing signal is transduced from the G protein to the promoters of penicillin biosynthesis genes. In the aflatoxin cluster of A. nidulans, the constitutively activated FadAG42R G subunit repressed expression of the aflR regulatory gene (encoding a positive transcriptional activator of the aflatoxin gene cluster), thus explaining the reduction of sterigmatocystin production by a cascade mechanism (Tag et al., 2000; Calvo et al., 2002).
The molecular cascade involved in expression of the three penicillin biosynthesis genes reported in this work is unknown since no regulatory gene of the zinc-finger family equivalent to AflR has been found in the penicillin gene cluster. A related gene has been reported in the 56.8 kb amplified region surrounding the penicillin gene cluster (Fierro et al., 2006), but it does not seem to play a relevant role in penicillin gene expression (van den Berg et al., 2007). In A. nidulans a zinc-finger regulator is located close to the penicillin gene cluster (Fierro et al., 2006), but its possible role as a signal transducer to regulate penicillin gene expression is unknown.
Pga1 regulates apical extension in solid medium (García-Rico et al., 2007), conidiation (García-Rico et al., 2008) and secondary metabolite production in P. chrysogenum (this work). cAMP is a secondary messenger in the Pga1-mediated signal transduction pathway, and its intracellular levels are clearly regulated by Pga1 (García-Rico et al., 2008). However, cAMP has a very different degree of involvement in processes regulated by Pga1. While cAMP has an important role in the regulation of apical extension of P. chrysogenum NRRL 1951 (R. O. García-Rico et al., unpublished results), it plays only a minor role in regulation of conidiation (García-Rico et al., 2008), and, as shown in this work, it seems to play no relevant role in penicillin production. The cAMP-dependent protein kinase PkaA is a downstream effector of FadA and has been implicated in repression of both conidiation and sterigmatocystin production in A. nidulans (Shimizu & Keller, 2001), but the authors proposed the existence of an alternative cAMP/PkaA-independent pathway for the regulation of conidiation by FadA. Our data suggest that different processes regulated by Pga1 are mediated by distinct cAMP-dependent or cAMP-independent pathways in P. chrysogenum.
Participation of cAMP in G protein signalling is complex, and it may act at different points in distinct pathways. Two other genes encoding G protein subunits are usually present in fungi regulating different processes; for instance in Gibberella zeae the GzGPA1 G subunit, a homologue of Pga1 and FadA and belonging to subgroup I of fungal G subunits (Bölker, 1998), controls sexual sporulation and represses toxin production, whereas GzGPA2 (belonging to subgroup III) regulates pathogenicity, and GzGPA3 (subgroup II) may participate in regulation of vegetative growth along with other subunits (Yu et al., 2008). cAMP has been proposed to mediate different processes regulated by G protein signalling; in addition to its role as downstream effector of FadA signalling in A. nidulans (Shimizu & Keller, 2001), the cAMP/PKA system is involved downstream of group III G-protein signalling in processes such as cell proliferation, development, stress response, mating and virulence, and proposed to mediate regulation of germination by GanB signalling in A. nidulans (Chang et al., 2004).
Mutations in the pga1 and G203R-T strains are not equivalent. Whereas in strain pga1 the β dimer of the heterotrimeric G protein is present in the cell in a free form and therefore active, in strain G203R-T the β dimer is titrated out upon binding to the mutant G subunit and consequently rendered inactive. The β dimer seems to play some role in conidiation, as the phenotype of these strains regarding conidiation in submerged cultures differs significantly (García-Rico et al., 2008). The fact that the phenotype of both strains regarding secondary metabolite production is very similar suggests that regulation of secondary metabolism is mainly or exclusively mediated by the G subunit.
Our results raise the possibility of targeting the G protein components for improved production of penicillin. This strategy may also serve to enhance the production of unknown secondary metabolites that are encoded by poorly expressed genes, as is the case for roquefortine in P. chrysogenum. Some of the clusters for secondary metabolites are considered to be silent because they are not expressed or are transcribed below detectable levels (Martín, 2000; Metsä-Ketelä et al., 2004). Altering the heterotrimeric G protein signalling may be useful to uncover new bioactive secondary metabolites from fungi.
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ACKNOWLEDGEMENTS |
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Edited by: S. D. Harris
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REFERENCES |
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Asilonu, E., Bucke, C. & Keshavarz, T. (2000). Enhancement of chrysogenin production in cultures of Penicillium chrysogenum by uronic acid oligosaccharides. Biotechnol Lett 22, 931–936.[CrossRef]
Bohm, A., Gaudet, R. & Sigler, P. B. (1997). Structural aspects of heterotrimeric G-protein signaling. Curr Opin Biotechnol 8, 480–487.[CrossRef][Medline]
Bölker, M. (1998). Sex and crime: heterotrimeric G proteins in fungal mating and pathogenesis. Fungal Genet Biol 25, 143–156.[CrossRef][Medline]
Brakhage, A. A. (1998). Molecular regulation of beta-lactam biosynthesis in filamentous fungi. Microbiol Mol Biol Rev 62, 547–585.
Calvo, A. M., Wilson, R. A., Bok, J. W. & Keller, N. P. (2002). Relationship between secondary metabolism and fungal development. Microbiol Mol Biol Rev 66, 447–459.
Casqueiro, J., Gutiérrez, S., Bañuelos, O., Hijarrubia, M. J. & Martín, J. F. (1999). Gene targeting in Penicillium chrysogenum: disruption of the lys2 gene leads to penicillin overproduction. J Bacteriol 181, 1181–1188.
Chang, M. H., Chae, K. S., Han, D. M. & Jahng, K. Y. (2004). The GanB G-protein negatively regulates asexual sporulation and plays a positive role in conidial germination in Aspergillus nidulans. Genetics 167, 1305–1315.
de la Campa, R., Seifert, K. & Miller, J. D. (2007). Toxins from strains of Penicillium chrysogenum isolated from buildings and other sources. Mycopathologia 163, 161–168.[CrossRef][Medline]
Elander, R. P. (2003). Industrial production of beta-lactam antibiotics. Appl Microbiol Biotechnol 61, 385–392.[Medline]
Fierro, F., Barredo, J. L., Díez, B., Gutiérrez, S., Fernández, F. J. & Martín, J. F. (1995). The penicillin gene cluster is amplified in tandem repeats linked by conserved hexanucleotide sequences. Proc Natl Acad Sci U S A 92, 6200–6204.
Fierro, F., Montenegro, E., Gutiérrez, S. & Martín, J. F. (1996). Mutants blocked in penicillin biosynthesis show a deletion of the entire penicillin gene cluster at a specific site within a conserved hexanucleotide sequence. Appl Microbiol Biotechnol 44, 597–604.[CrossRef][Medline]
Fierro, F., Martín, J. F. & Kosalková, K. (2002). Sulfur-containing β-lactam antibiotics: enzymes, genes and regulation of the biosynthesis. In Microbial Secondary Metabolites: Biosynthesis, Genetics and Regulation, pp. 179–210. Edited by F. Fierro & J. F. Martín. Trivandrum, India: Research Signpost.
Fierro, F., García-Estrada, C., Castillo, I., Rodríguez, R., Velasco-Conde, T. & Martín, J. F. (2006). Transcriptional and bioinformatic analysis of the 56.8 kb DNA region amplified in tandem repeats containing the penicillin gene cluster in Penicillium chrysogenum. Fungal Genet Biol 43, 618–629.[CrossRef][Medline]
Frisvad, J. C. & Filtenborg, O. (1983). Classification of terverticillate penicillia based on profiles of mycotoxins and other secondary metabolites. Appl Environ Microbiol 46, 1301–1310.
Galagan, J. E., Calvo, S. E., Cuomo, C., Ma, L. J., Wortman, J. R., Batzoglou, S., Lee, S. I., Ba
türkmen, M., Spevak, C. C. & other authors (2005). Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae. Nature 438, 1105–1115.[CrossRef][Medline]
García-Rico, R. O., Martín, J. F. & Fierro, F. (2007). The pga1 gene of Penicillium chrysogenum encodes a heterotrimeric G protein alpha subunit that controls growth and development. Res Microbiol 158, 437–446.[Medline]
García-Rico, R. O., Fierro, F. & Martín, J. F. (2008). Heterotrimeric G protein Pga1 of Penicillium chrysogenum controls conidiation mainly by a cAMP-independent mechanism. Biochem Cell Biol 86, 57–69.[CrossRef][Medline]
Gutiérrez, S., Marcos, T., Casqueiro, J., Kosalkovà, K., Fernández, F., Velasco, J. & Martín, J. F. (1999). Transcription of the pcbAB, pcbC and penDE genes of Penicillium chrysogenum AS-P-78 is repressed by glucose and the repression is not reversed by alkaline pHs. Microbiology 145, 317–324.
Hamm, H. E. (1998). The many faces of G protein signaling. J Biol Chem 273, 669–672.
Hicks, J. K., Yu, J. H., Keller, N. P. & Adams, T. H. (1997). Aspergillus sporulation and mycotoxin production both require inactivation of the FadA G alpha protein-dependent signaling pathway. EMBO J 16, 4916–4923.[CrossRef][Medline]
Hoffmeister, D. & Keller, N. P. (2007). Natural products of filamentous fungi: enzymes, genes, and their regulation. Nat Prod Rep 24, 393–416.[CrossRef][Medline]
Keller, N. P., Turner, G. & Bennett, J. W. (2005). Fungal secondary metabolism – from biochemistry to genomics. Nat Rev Microbiol 3, 937–947.[CrossRef][Medline]
Kosalkova, K., Marcos, A. T., Fierro, F., Hernando-Rico, V., Gutiérrez, S. & Martín, J. F. (2000). A novel heptameric sequence (TTAGTAA) is the binding site for a protein required for high level expression of pcbAB, the first gene of the penicillin biosynthesis in Penicillium chrysogenum. J Biol Chem 275, 2423–2430.
Kurjan, J. (1992). Pheromone response in yeast. Annu Rev Biochem 61, 1097–1129.[CrossRef][Medline]
Lengeler, K. B., Davidson, R. C., d'Souza, C., Harashima, T., Shen, W. C., Wang, P., Pan, X., Waugh, M. & Heitman, J. (2000). Signal transduction cascades regulating fungal development and virulence. Microbiol Mol Biol Rev 64, 746–785.
Li, L., Wright, S. J., Krystofova, S., Park, G. & Borkovich, K. A. (2007). Heterotrimeric G protein signaling in filamentous fungi. Annu Rev Microbiol 61, 423–452.[CrossRef][Medline]
Liras, P. & Martín, J. F. (2006). Gene clusters for β-lactam antibiotics and control of their expression: why have clusters been formed and where do they come from? Int Microbiol 9, 9–19.[Medline]
Machida, M., Asai, K., Sano, M., Tanaka, T., Kumagai, T., Terai, G., Kusumoto, K., Arima, T., Akita, O. & other authors (2005). Genome sequencing and analysis of Aspergillus oryzae. Nature 438, 1157–1161.[CrossRef][Medline]
Martín, J. F. (1998). New aspects of genes and enzymes for β-lactam antibiotic biosynthesis. Appl Microbiol Biotechnol 50, 1–15.[CrossRef][Medline]
Martín, J. F. (2000). Molecular control of expression of penicillin biosynthesis genes in fungi: regulatory proteins interact with a bidirectional promoter region. J Bacteriol 182, 2355–2362.
Metsä-Ketelä, M., Ylihonko, K. & Mäntsälä, P. (2004). Partial activation of a silent angucycline-type gene cluster from a rubromycin beta producing Streptomyces sp. PGA64. J Antibiot (Tokyo) 57, 502–510.[Medline]
Neer, E. J. (1995). Heterotrimeric G proteins: organizers of transmembrane signals. Cell 80, 249–257.[CrossRef][Medline]
Nielsen, K. F. & Smedsgaard, J. (2003). Fungal metabolite screening: database of 474 mycotoxins and fungal metabolites for dereplication by standardised liquid chromatography-UV-mass spectrometry methodology. J Chromatogr A 1002, 111–136.[CrossRef][Medline]
Nierman, W. C., Pain, A., Anderson, M. J., Wortman, J. R., Kim, H. S., Arroyo, J., Berriman, M., Abe, K., Archer, D. B. & other authors (2005). Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus. Nature 438, 1151–1156.[CrossRef][Medline]
Reithner, B., Brunner, A., Schuhmacher, R., Peissl, I., Seidl, V., Krska, R. & Zeilinger, S. (2005). The G protein subunit Tga1 of Trichoderma atroviride is involved in chitinase formation and differential production of antifungal metabolites. Fungal Genet Biol 42, 749–760.[CrossRef][Medline]
Rundberget, T., Skaar, I. & Flaoyen, A. (2004). The presence of Penicillium and Penicillium mycotoxins in food wastes. Int J Food Microbiol 90, 181–188.[CrossRef][Medline]
Scott, P. M., Lawrence, J. W. & van Walbeek, W. (1970). Detection of mycotoxins by thin-layer chromatography: application to screening of fungal extracts. Appl Microbiol 20, 839–842.[Medline]
Shimizu, K. & Keller, N. P. (2001). Genetic involvement of a cAMP-dependent protein kinase in a G protein signaling pathway regulating morphological and chemical transitions in Aspergillus nidulans. Genetics 15, 591–600.
Suárez, T. & Peñalva, M. A. (1996). Characterisation of a Penicillium chrysogenum gene encoding a PacC transcription factor and its binding sites in the divergent pcbAB-pcbC promoter of the penicillin biosynthetic cluster. Mol Microbiol 20, 529–540.[CrossRef][Medline]
Tag, A., Hicks, J., Garifullina, G., Ake, C., Jr, Phillips, T. D., Beremand, M. & Keller, N. (2000). G-protein signalling mediates differential production of toxic secondary metabolites. Mol Microbiol 38, 658–665.[CrossRef][Medline]
van den Berg, M. A., Westerlaken, I., Leeflang, C., Kerkman, R. & Bovenberg, R. A. (2007). Functional characterization of the penicillin biosynthetic gene cluster of Penicillium chrysogenum Wisconsin54-1255. Fungal Genet Biol 44, 830–844.[CrossRef][Medline]
Yang, Q. & Borkovich, K. A. (1999). Mutational activation of a Gi causes uncontrolled proliferation of aerial hyphae and increased sensitivity to heat and oxidative stress in Neurospora crassa. Genetics 151, 107–117.
Yu, J. H. & Keller, N. (2005). Regulation of secondary metabolism in filamentous fungi. Annu Rev Phytopathol 43, 437–458.[CrossRef][Medline]
Yu, J. H., Wieser, J. & Adams, T. H. (1996). The Aspergillus FlbA RGS domain protein antagonizes G protein signaling to block proliferation and allow development. EMBO J 15, 5184–5190.[Medline]
Yu, H.-Y., Seo, J.-A., Kim, J.-E., Han, K.-H., Shim, W.-B., Yun, S.-H. & Lee, Y.-W. (2008). Functional analyses of heterotrimeric G protein G and Gβ subunits in Gibberella zeae. Microbiology 154, 392–401.
Zhang, Y. Q., Wilkinson, H., Keller, N. P. & Tsitsigiannis, D. I. (2004). Secondary metabolite gene clusters. In Handbook of Industrial Microbiology, pp. 355–386. Edited by Z. An. New York: Marcel Dekker.
Zuber, S., Hynes, M. J. & Andrianopoulos, A. (2002). G-protein signaling mediates asexual development at 25 °C but has no effect on yeast-like growth at 37 °C in the dimorphic fungus Penicillium marneffei. Eukaryot Cell 1, 440–447.
Received 3 April 2008;
revised 9 July 2008;
accepted 11 July 2008.
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, Repression; +, activation. Pga1 negatively regulates conidiation by repressing expression of genes brlA and wetA, which belong to the conidiation central regulatory pathway (García-Rico et al., 2008