Effects of mutations in the GanB/RgsA G protein mediated signalling on the autolysis of Aspergillus...

J. Basic Microbiol. 46 (2006) 6, 495–503 DOI: 10.1002/jobm.200610174

© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 0233-111X/06/0612-0495

(Department of Microbiology and Biotechnology, Faculty of Science, University of Debrecen, H-4010 Debrecen, P.O. Box: 63, Hungary)

Effects of mutations in the GanB/RgsA G protein mediated signalling on the autolysis of Aspergillus nidulans

ZSOLT MOLNÁR, TAMÁS EMRI*, ERZSÉBET ZAVACZKI, TÜNDE PUSZTAHELYI and ISTVÁN PÓCSI

(Received 08 March 2006/Returned for modification 27 March 2006/Accepted 28 April 2006)

Physiological changes taking place in carbon-starved, autolysing cultures of Aspergillus (Emericella) nidulans strains with mutations in the GanB/RgsA heterotrimeric G protein signalling pathway were studied and compared. Deletion of the ganB, rgsA or both genes did not alter markedly either the autolytic loss of biomass or the extracellular chitinase production. However, they caused a significant decrease in the proteinase formation, which was detected by measuring both extracellular enzyme activity and the transcription of the prtA gene. The deletion mutants also showed significantly higher specific γ-glutamyltranspeptidase activities than the control strain. Deletion of the rgsA gene affected the glutathione peroxidase and catalase formation, as well as the peroxide content of the cells. The concomitant initiations of cell death and developmental genomic programmes may be interconnected via heterotrimeric G-protein signalling and subsequent changes in intracellular ROS levels in ageing A. nidulans. G protein mediated signalling pathways have a crucial role in the regulation of asexual sporulation, vegetative growth and sense of various extracellular signals in Aspergillus nidulans (ADAMS et al. 1998, CHANG et al. 2004). In the A. nidulans genome nine putative GPCR coding genes (gprA–L) were identified (HAN et al. 2004, SEO et al. 2004). The suit-able G protein pairs of the GPCRs have not been identified yet despite of that in A. nidulans there are three known G protein α subunits (FadA, GanA-B) (LEE and ADAMS 1994, YU et al. 1996, CHANG et al. 2004, HAN et al. 2004, LAFON et al. 2005). Heterotrimeric G pro-teins are controlled by RGS (regulator of G protein signalling) proteins, of which four (flbA, rgsA-C) have been found by this time (LEE and ADAMS 1994, YU et al. 1996, 1999, WIESER et al. 1997, HAN et al. 2004). The RGS pair of FadA is FlbA (LEE et al. 1994, YU et al. 1996, 1999) while GanB is controlled by RgsA (HAN et al. 2004). Since there is only one Gβ and Gγ subunit in the A. nidulans genome database (CHANG et al. 2004) the FadA/FlbA and GanB/RgsA pathways seem to share the same Gβγ subunit. The FadA/FlbA signalling maintains vegetative growth and represses asexual and sexual development as well as sterigmatocystin production (LEE et al. 1994, YU et al. 1996, 1999, ADAMS et al. 1998, SEO et al. 2005). The GanB/RgsA signalling plays a positive role during germination of conidia and ascospores through carbon source sensing and negatively regulates asexual sporulation (CHANG et al. 2004, HAN et al. 2004, LAFON et al. 2005). It also controls pigment synthesis and some aspects of stress responses in A. nidulans (HAN et al. 2004). Here we present the role of GanB/RgsA signalling (CHANG et al. 2004, HAN et al. 2004, LAFON et al. 2005) in the carbon starvation induced stress response of Aspergillus nidulans.

* Corresponding author: Prof. T. EMRI; e-mail: [email protected]

496 Z. MOLNÁR et al.

© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

Materials and methods

Aspergillus nidulans (Emericella nidulans) RKH51.117 (pabaA1, yA2), RKH51.09 (pabaA1, yA2, ∆rgsA::argB+), RMdgB03 (pabaA1, yA2; ∆argB::trpC+ ∆ganB::argB+, trpC801) and RKH52.02 (pabaA1, yA2, ∆rgsA::argB+, ∆ganB:: argB+) strains were the kind gift of Dr. JAE-HYUK YU (University of Wisconsin-Madison, USA). Strains were grown in shake flasks (500 ml) containing 100 ml minimal-nitrate medium (pH 6.5) supplemented with 0.5% yeast extract and with 20 µg l–1 4-aminobenzoic acid (YGL) (BARRATT et al. 1965). Culture media were inoculated with 5 × 107 spores and were incubated at 37 °C, 200 rpm for up to 168 h. Cell viability was tested by transferring samples, taken at different cultivation times, into fresh media and measuring gains in dry cell mass (DCM). For glutathione (GSH) and glutathione disulphide (GSSG) determinations, mycelia were separated from 10 ml aliquots of the cultures by filtration on sintered glass. Cells were washed with distilled water and were re-suspended in ice-cold 5% (w/v) 5-sulfosalicylic acid by vigorous mixing and were left at 0 °C for 20 min (EMRI et al. 1999). After centrifugation at 10,000 g for 10 min, the supernatants were neutralised with triethanolamine at 0 °C. The intracellular GSH and GSSG concentrations were determined according to Anderson (1985). The intracellular peroxide and superoxide levels were characterised in separate experiments by the formation of 2′,7′-dichlorofluorescein (DCF) from 2′,7′-dichlorofluorescin diacetate and ethidium (Et) from dihydroethidium, respectively, as described earlier (SÁMI et al. 2001a). Changes in the specific activities of certain intracellular enzymes were also followed in separate experiments. In these cases, mycelia were harvested by filtration on sintered glass were washed by distilled water and were resuspended in ice-cold 0.1 M K-phosphate buffer (pH 7.5). Cell-free extracts were prepared by X-pressing and centrifugation (EMRI et al. 1999). Specific glutathione peroxidase (GPx) (CHIU et al. 1976), γ-glutamyltranspeptidase (γGT) (EMRI et al. 1997) and catalase (ROGGEN-KAMP et al. 1974) activities were measured by the methods indicated in parentheses. The glucose consumption was measured by the rate assay of LEARY et al. (1992). Extracellular proteinase activities of the filtrates were characterised by the velocity constant of the enzyme reaction (K) according to TOMARELLI et al. (1949). Extracellular chitinase activities were determined using CM-chitin-RBV (LOEWE Biochimica GmbH, Sauerlach, Germany) as substrate (EMRI et al. 2004b). DCM of the samples was determined as described in previous publications (PUSZTAHELYI et al. 1997a,b) and protein contents of the cell-free extracts were measured by a modification of the LOWRY method (1983). Osmotic stress tolerance of the rgsA and ganB mutant and the RKH51.117 control strains was cha-racterized by minimal inhibitory concentrations (MICs) of NaCl determined on solid minimal-nitrate medium (pH 6.5) supplemented with 20 µg l–1 4-aminobenzoic acid (BARRATT et al. 1965) and also containing 0, 1, 1.5, 2, 2.5 or 3 mol l–1 NaCl. Agar plates were incubated at 37 °C for 48 h, and all experiments were performed in triplicates. MICs were defined as the lowest NaCl concentrations, which completely inhibited the growth of the strains tested. Total RNA was extracted from freeze-dried mycelial mats with TRISOL reagent (Invitrogen, LOFER, Austria) as recommended by CHOMCZYNSKI (1993). RNA was DNAse treated and used in RT-PCR mRNA quantification assays. RNA samples (7.5 µg) were run in 1% agarose gel for quality control. Only RNA preparations with same intensity rRNA bands and no clue of RNA degradation were applied in RT-PCR reactions. In ageing cultures, RNA samples with no degradation or smears in rRNA bands could only be extracted up to 62 h of growth. RT-PCR reactions using QuantiTectTMSYBR®Green RT-PCR Kit (Qiagen, HILDEN, Germany) were carried out according to the manufacturer recommendations with 400 ng of total RNA per reaction, 2.5 mM of Mg2+ and 0.5 µM gene specific primers. The steps for the RT-PCR reaction were as follows: (1) reverse transcription, 50 °C, 30 min; (2) PCR initial activation step, 95 °C, 15 min; (3) DNA denaturation, 94 °C, 15 sec; (4) annealing, Tm – (5–8) °C, 30 sec; (4) extension, 72 °C, 30 sec and 40 cycles. The following oligonucleotide PCR primers were employed through this study to amplify specific gene transcripts: catA F: 5′-CAAACGCTCCGCCATCTAC-3′ and R: 5′-CTTGAGGTGCCCGAATGTC-3′; catB F: 5′-CCGAGCCCGACAACACTTAC-3′ and R: 5′-GTTCAGCGACGACAATGACG-3′; catC F: 5′-CAGAGCAAGCCGAGAAGTTC-3′ and R: 5′-CAAGGTGGGAGGGAGAGAAG-3′; prtA F: 5′-TTCTGTCCGTCAAGGTTTTC-3′ and R: 5′-TGAAGGCGTAAGAGTATCCAC-3′; prtB F: 5′-GCTTGAATCTCCTCTGTTTGC-3′ and R: 5′-GTCCAACCACCGTAGAAGAAG-3′.

Autolysis of Aspergillus nidulans 497


To determine the homogeneity of the RT-PCR products, the melting temperature was determined by heating up the products up to 95 °C and decreasing the temperature of the PCR system block stepwise to 55 °C by 0.5 °C, meanwhile the fluorescence was read at each step. The same PCR products were also subjected to 1% agarose gel-electrophoresis in 1 × TAE buffer. All cDNA amplification products evaluated and presented in this paper were found homogeneous by both methods. The imprecision of the RT-PCR assay was evaluated for each gene tested. Within run CV values were always less than 4.1% (n = 8) while between run CV values were maximum 5% (n = 5). Standard deviations (SD) were determined for each gene and culture condition tested, and SD values were always less than 1 cycles. SD values were also calculated for all other assays employed in this study to estimate variations between experiments, and the statistical significance of changes in physiological parameters was estimated by the Student’s t-test. Only the probability levels of P ≤ 5% were regarded as indicative of the statistical significance. Unless otherwise indicated, all the chemicals were purchased from the SIGMA-ALDRICH Ltd., Buda-pest, Hungary.

Results

Autolytic process induced by carbon starvation of the RKH51.09 (∆rgsA), RMdgB03 (∆ganB), RKH52.02 (∆rgsA∆ganB) mutants and the RKH51.117 control strain was investi-gated in YGL medium. Glucose was completely exhausted by 24 h of cultivation in all cul-

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Fig. 1 Changes in the DCM (A), extracellular chitinase (B) and proteinase (C) activities during autolysis of Aspergillus nidulans RKH51.09 (∆rgsA; �), RMdgB03 (∆ganB; �), RKH52.02 (∆rgsA∆ganB, �) and RKH51.117 (control; �) strains. Symbols represent means calculated from 4 independent experi-ments. The S.D. values were less than 15%



tures (data not shown) and the autolytic process started thereafter (Fig. 1; MOLNÁR et al. 2004). As can be seen in Fig. 1A–B, the lack of rgsA and/or ganB genes caused only minor differences in the extracellular chitinase activities and the autolytic declination of the DCM. In contrast, significant decreases in the extracellular proteinase activities were detected during autolysis in the mutant strains in proportion to the control (Fig. 1C). The proteinase activity in the ∆ganB mutant was as low as in the ∆rgsA∆ganB double mutant, while the deletion in the rgsA gene caused a lower decrease in the proteinase activity (Fig. 1C). The specific γGT activities were significantly higher in the ∆ganB and ∆rgsA mutants as well as in the ∆rgsA∆ganB double mutant than in the control strain (Fig. 2A). Interestingly, the observed changes in the γGT production did not result in a faster decrease in the GSH levels during autolysis (Fig. 2B) and did not cause significant changes in the GSSG concen-trations and in the GSH/GSSG ratio in comparison to the control (data not shown; MOLNÁR et al. 2004, EMRI et al. 2004a). The specific catalase activity of the control strain and the ∆ganB mutant were very simi-lar to each other in the whole period of observation (Fig. 3A). In the case of the ∆rgsA mu-tant, the catalase activities decreased only slowly during autolysis and, as a consequence, they were always higher than their counterparts observed in the control cultures starting from 50 h cultivation time (Fig. 3A). The double mutant showed an intermediate behaviour since the catalase activity of this strain started to decline 25 h later than that found in the control strain (Fig. 3A). On the other hand, the specific GPx activities of the ∆rgsA mutant were significantly lower than those found in the control strain up to 50 h incubation time while, in the two other mutants, the GPx production was higher than the control values (Fig. 3B). The accumulations of intracellular peroxides and superoxide followed very similar pat-terns in the four strains tested (Fig. 4A–B). Significantly reduced peroxide contents were only detected in the ∆rgsA mutant (Fig. 4A–B). The lower intracellular peroxide level found in the ∆rgsA mutant was not accompanied by a better viability in ageing cultures. After transferring harvested mycelia into fresh media, all the strains started to grow with very similar kinetics at all the tested time points independently of mutations and intracellu-lar peroxide levels (Fig. 4C). Deletion of the rgsA, ganB or both genes did not affect the osmotic stress tolerance of A. nidulans in the presence of NaCl. The MICNaCl values were 3 mol l–1 for all the strains tested independently of their genotypes.

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Fig. 2 Changes in the specific γGT activities (A) as well as in the GSH levels (B) during autolysis of Aspergillus nidulans RKH51.09 (∆rgsA; �), RMdgB03 (∆ganB; �), RKH52.02 (∆rgsA∆ganB, �) and RKH51.117 (control; �) strains. Symbols represent mean values calculated from 4 independent experiments. S.D. values were less than 15%



In order to determine which genes were responsible for the observed changes in the pro-teinase and catalase productions during autolysis we monitored the changes in the transcrip-tion of the catA-C (KAWASAKI et al. 1997, 2001) and prtA-B (KATZ et al. 1994, VANKUYK et al. 2000) genes by RT-PCR. We found that the expression of catA and catC was constitu-tive during the first 60 h of cultivations (data not shown). In the case of the catB gene, an induction-repression pattern with a maximum expression rate at 20 h cultivation time was recorded (Fig. 5A). Unfortunately, the quality of the RNA preparations declined fast after 60 h cultivation time and, therefore, we have no reliable transcriptional data in our hands when autolysis was progressing and the catalase activity remained high up to 168 h cultiva-tion time in the ∆rgsA mutant (Fig. 3A). The transcription of the prtA gene showed clear-cut down-regulation in the mutants (Fig. 5B) meanwhile the expression of prtB was constitutive in the whole period of observa-tion and was not responsive to mutations in the GanB/RgsA signalling pathway (data not shown).

Discussion

Carbon depleted autolysing A. nidulans cultures were characterized with a series of com- plex morphological and physiological changes (EMRI et al. 2004a, MOLNÁR et al 2004, Figs. 1–5). Glucose depletion triggered the production of extracellular chitinase and pro-teinase activities, concomitant fragmentation of hyphae, and decreases in DCM and pellet diameters (EMRI et al. 2004a, MOLNÁR et al. 2004, Fig. 1). Although the respiration de-creased markedly, carbon starvation increased the reactive oxygen species (ROS) contents of the cells and, as a consequence, induced antioxidant enzymes (EMRI et al. 2004a, Figs. 3---5). The viability of the cultures decreased and the development of apoptotic markers were detected (EMRI et al. 2005a). The specific activity of the GSH decomposing enzyme γGT also increased and, hence, the GSH pool shrank considerably (EMRI et al. 2004a, Fig. 2). The FluG-BrlA pathway, one of the most important pathways that regulate the bal-ance between sporulation and vegetative growth in A. nidulans (ADAMS et al. 1998), was essential in the induction of autolysis (EMRI et al. 2005b). It induced the extracellular hy-drolase production, DCM declination and hyphal fragmentation as well (EMRI et al. 2005b).

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Fig. 3 Changes in the specific catalase (A) and GPx (B) activities during the autolysis of Aspergillus nidulans RKH51.09 (∆rgsA; �), RMdgB03 (∆ganB; �), RKH52.02 (∆rgsA∆ganB, �) and RKH51.117 (control; �) strains. Symbols represent mean values calculated from 4 independent experiments. S.D. values were less than 10%



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Fig. 4 Age-dependent alterations in the production of peroxides (DCF; Part A) and superoxide (Et; Part B) in Aspergillus nidulans RKH51.09 (∆rgsA; �), RMdgB03 (∆ganB; �), RKH52.02 (∆rgsA∆ganB, �) and RKH51.117 (control; �) strains. Part C shows the growth kinetics of the same strains after transferring their 11 d old cultures into fresh YGL medium. Similar results were found when mycelia were transferred into fresh medium on the 5th or 7th day of cultivation (data not shown). Symbols represent mean values calculated from 3 independent experiments. S.D. values were less than 17% (Parts A and B) or 11%. (Part C)

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Fig. 5 Age-dependent changes in the expression levels of the catB (A) and prtA (B) genes in Aspergil- lus nidulans RKH51.09 (∆rgsA; �), RMdgB03 (∆ganB; �), RKH52.02 (∆rgsA∆ganB, �) and RKH51.117 (control; �) strains. Symbols represent mean values calculated from 3 independent experiments. S.D. values were less than 12%



Interestingly, mutations in the pathway did not affect the apoptosis-like cell death during carbon starvation (EMRI et al. 2005a). On the other hand, heterotrimeric G proteins – which are also involved in the regulation of sporulation and vegetative growth (YU et al. 1996, 1999, CHANG et al. 2004, HAN et al. 2004) – had only minor effect on the autolytic process itself. E.g. their mutants did not cause significant changes in the DCM declination and chitinase production (MOLNÁR et al. 2004; Fig. 1). However, the FadA/FlbA signalling controlled the fragmentation of hyphae (MOLNÁR et al. 2004), and together with the GanB/ RgsA pathway also regulated the proteinase production (MOLNÁR et al. 2004; Fig. 1C, Fig. 5B). Interest-ingly, while mutations in the FadA/FlbA signalling pathway caused only a time shift in the proteinase formation (MOLNÁR et al. 2004), the GanB/RgsA mediated signalling affected the specific proteinase production (Fig. 1C). In the case of GanB/RgsA, we also demon-strated that, between the two main extracellular proteinases of A. nidulans (VAN KUYK et al. 2000), this pathway regulated prtA and had no effect on the expression of prtB (Fig. 5B). The ammonia production of carbon starving cultures was indicative of the utilisation of organic nitrogen compounds as energy sources (EMRI et al. 2004a). Similar to proteins, the high GSH content of the cells can also be an alternative nutrient for carbon starving cultures (EMRI et al. 2004a). Both heterotrimeric G protein-dependent signal transduction pathways affected the specific activity of γGT, the main GSH degrading enzyme (MOLNÁR et al. 2004; Fig 2). The time shift caused by FadA/FlbA mutations in the induction of γGT re-sulted in parallel time shifts in the age-dependent GSH degradation (MOLNÁR et al. 2004). However, the increased γGT activities observed in ganB and rgsA deletion mutants did not facilitate the degradation of GSH during autolysis, which is usually a fast metabolic process triggered by carbon depletion (MOLNÁR et al. 2004; Fig. 2). Considering the effects of the FadA/FlbA and GanB/RgsA heterotrimeric G protein signalling pathways on GSH metabo-lism and proteinase formation, we can assume that, besides the regulation of asexual repro-duction, one of the main physiological functions of these signals is to control the mobilisa-tion of available organic nitrogen storage compounds as carbon and energy sources in carbon-depleted cultures. In carbon-starved cultures, continuous accumulation of ROS, induction of SOD and tem-poral induction of GPx and catalase, namely the catB gene (Fig. 5A) were observed (EMRI et al. 2004a, b, MOLNÁR et al. 2004; Figs. 3–4). HAN et al. (2004) demonstrated that RgsA was involved in protection against both oxidative stress and heat stress since deletion of the rgsA gene resulted in an enhanced H2O2 and heat tolerance. However, mutations in the GanB/RgsA pathway did not affect the osmotolerance of the fungus. In our cultures, RgsA influenced the specific GPx (Fig. 3B) and catalase (Fig. 3A) activities as well as the perox-ide content of the cells (Fig. 4A). However, these changes did not result in prolonged viabil-ity at all (Fig. 4C). It is worth underlining that despite of accumulating ROS, autolysing cells increased the γGT specific activity, and, as a consequence, decreased intracellular GSH concentrations under the dual control of the FadA/FlbA and GanB/RgsA pathways. Mean-while, the specific catalase was also reduced via GanB/RgsA signalling. Although the intracellular accumulation of ROS may cause oxidative cell damages and facilitate the progression of ageing and programmed cell death (LEITER et al. 2005) this may also initiate sporulation (EMRI et al. 2004b, AGUIRRE et al. 2005). Therefore, both cell death and developmental genomic programmes may be initiated and coupled via heterotrimeric G-protein signalling pathways and their influence on intracellular ROS levels in A. nidulans (EMRI et al. 2004b, AGUIRRE et al. 2005, LEITER et al. 2005).

Acknowledgements

The Hungarian Ministry of Education awarded a Széchenyi Scholarship for Professors to I.P., and T.E. was a grantee of the Bolyai János Scholarship.



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Mailing address: TAMÁS EMRI, Department of Microbiology and Biotechnology, Faculty of Sciences, University of Debrecen, P.O. Box: 63, H-4010 Debrecen, Hungary E-mail: [email protected]

Effects of mutations in the GanB/RgsA G protein mediated signalling on the autolysis of Aspergillus...

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