Disruption of the gene encoding the V-ATPase subunit A results in inhibition of normal growth and abolished sporulation in Aspergillus nidulans

Petter Melin1, Johan Schnürer1 and E. Gerhart H. Wagner2

1 Department of Microbiology, Swedish University of Agricultural Sciences, Box 7025, S-750 07 Uppsala, Sweden
2 Institute of Cell and Molecular Biology, Department of Microbiology, Biomedical Center, Uppsala University, Box 596, S-75124 Uppsala, Sweden

Correspondence
Petter Melin
petter.melin{at}mikrob.slu.se


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
The authors have previously reported on molecular responses of Aspergillus nidulans to bacterial antifungal metabolites, e.g. bafilomycins and the related concanamycins. These compounds are known inhibitors of V-ATPases and cause dramatic effects on mycelial growth and morphology. In Neurospora crassa, studies have shown that disruption of the gene encoding subunit A of the V-ATPase results in morphological changes and reduced growth similar to those observed after addition of concanamycin. This phenotype, and the fact that this mutation confers resistance to concanamycin, suggests that V-ATPase is the main (or only) target for the antibiotics. However, growth inhibition and morphology changes in, for example, A. nidulans and Penicillium roqueforti are more severe, and thus other targets are possible. In this study, the vmaA gene of A. nidulans, encoding the subunit A of V-ATPase, was disrupted by homologous recombination. The resulting vmaA1 mutant strain displayed extremely slow growth and failed to produce asexual spores. Furthermore, an altered morphology similar to that caused by addition of V-ATPase inhibitors, i.e. bafilomycin or concanamycin, was observed, indicating that V-ATPase is the main target for the antibiotics also in A. nidulans. The vmaA1 mutant was not viable at pH values above 7 and was highly sensitive to high Zn2+ concentrations, in agreement with previous results from studies of Saccharomyces cerevisiae and N. crassa.


The EMBL/Genbank accession number for the new sequence in this paper is AJ511279.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
The fungal vacuole is involved in several functions, e.g. macromolecular degradation, storage, and regulation of metabolites and ions (Klionsky et al., 1990; Weber, 2002). The pH of fungal vacuoles is regulated by vacuolar ATPases (V-ATPases) working as proton pumps. V-ATPases are present in all eukaryotes, in vacuoles in fungi and plants, and in several animal organelles such as endosomes and lysosomes (Forgac, 1989).

In a previous study we identified a strain of Streptomyces halstedii which produces compounds that both reduce the growth and dramatically alter the morphology of filamentous fungi. This effect was severe in all filamentous fungi tested but varied between species, e.g. Penicillium roqueforti is more sensitive than Aspergillus fumigatus (Frändberg & Schnürer, 1998). These substances were identified as bafilomycin B1 and bafilomycin C1 (Frändberg et al., 2000). The bafilomycins, and the related concanamycins, are known inhibitors of V-ATPases at nanomolar concentrations (Bowman et al., 1988; Dröse et al., 1993). Studies in Neurospora crassa showed that a similar growth inhibition and morphology change triggered by concanamycin treatment was obtained after disruption of vma-1, the gene encoding subunit A of the V-ATPase. These mutant strains were also resistant to concanamycin, suggesting that the V-ATPase is the only target for the antibiotic (Bowman et al., 2000). The inhibitory effect caused by bafilomycins is more severe in Aspergillus than in Neurospora (Werner & Hagenmaier, 1984). A further increase in antibiotic concentration results in increased swelling of Aspergillus mycelium, and growth is almost completely inhibited (unpublished observations). This indicates that a disruption of the Aspergillus V-ATPase gene results in a more severe phenotype. Alternatively, there may be additional, so far unidentified, targets for the antibiotics. Thus, it has been reported that bafilomycin is active against P-ATPases at micromolar concentrations (Bowman et al., 1988).

To investigate fungal responses to these antibiotics, two previous studies have employed mRNA differential display and proteomics approaches, respectively, to search for genes and proteins in A. nidulans involved in the response (Melin et al., 1999, 2002). These studies have so far only identified a small number of genes/gene products. One of these, breA (down-regulated in response to bafilomycin treatment), had previously been identified as aspnd1, encoding a cell-wall-associated zinc-binding protein involved in zinc uptake into the cytoplasm (Segurado et al., 1999).

The objective of this study was to investigate the V-ATPase of Aspergillus, in particular with respect to the question whether additional targets for bafilomycins and concanamycins could be present. Here, we report the phenotypic effects of a disruption of the gene encoding one of the subunits of the A. nidulans V-ATPase. We anticipate that the construction of this mutant strain will be instrumental in further studies of the previously identified gene products whose abundance is affected by antibiotic treatment of the fungus.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Growth conditions.
The A. nidulans strains used in this study are summarized in Table 1. Strains were cultured on minimal medium with supplements as described by Kaminskyj (2001). A. nidulans genetic methods followed standard protocols (Pontecorvo et al., 1953). For cloning and plasmid propagation Escherichia coli strain DH5{alpha} was used (Sambrook et al., 1989).


View this table:
[in this window]
[in a new window]
 
Table 1. A. nidulans strains used in this study

 
Preparation of DNA.
The procedure for growing and maintaining strains for DNA preparation varied. To extract DNA from the vmaA1 mutant strains (PM6 and PM7), fractions of the colony were spread on minimal plates covered with cellophane. DNA from the parental strains was extracted from submerged cultures. Mycelia were freeze-dried overnight. The pellet-like tissue was crushed using a toothpick, and DNA was extracted with CTAB as described by Talbot (2001). Prior to digestion, the genomic DNA was purified by phenol/chloroform extraction.

Cloning and disruption of the vmaA gene.
A 2 kb fragment containing the vmaA gene was PCR-amplified (forward primer 5'-GTA TGG ATC CCA CTG TGG GA-3'; reverse primer, with an introduced XbaI site, 5'-CTC TTC TAG ACG ACC AGA AAG CT-3') using pfu turbo polymerase (Stratagene). The DNA fragments were digested with BamHI and XbaI and subcloned into a modified (disrupted XhoI and SalI sites) pBS SK+ vector plasmid (Stratagene). Next, the construct was digested with XhoI to replace a 112 bp fragment, which includes the translation initiation codon, by the complete A. nidulans argB gene. The vmaA disruption construct was transformed as previously described (Melin et al., 2003). DNA used for transformation was PCR-amplified (primers as above) using taq+ polymerase (Stratagene) and purified with QiaQuick (Qiagen). The gene disruption was confirmed by Southern blot analysis, using a vmaA-specific probe which was 32P-labelled by random priming, according to standard methods (Fig. 1; Sambrook et al., 1989).



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 1. Targeted gene disruption of vmaA using a linear 3·6 kb fragment. The argB gene replaces a 111 bp XhoI fragment of vmaA after a double-crossover recombination event. Shaded areas represent regions of sequence identity used in recombination. Relevant restriction sites, and the location of the probe used for Southern hybridization, are indicated.

 
Viability tests.
Tests for sensitivity to bafilomycin B1 and concanamycin A were performed as previously described (Melin et al., 2002), with the exception that ascospores (equivalent to approximately 200 c.f.u. ml-1) were used instead of conidia. To test the viability of the vmaA1 strain, ascospores (2000 c.f.u. ml-1) from the cross between vmaA1 and A851 were plated on minimal plates containing various additions, supplemented with p-aminobenzoic acid, and incubated at 30 °C. To increase the pH, plates were buffered with 0·2 M K2HPO4. To decrease the pH, the plates were buffered with 20 mM MES. Zn2+ concentrations were raised from the standard 0·15 mM by adding ZnSO4. To increase the standard iron concentration (3·6 nM Fe2+) we used either FeSO4 or Fe2(SO4)3.


   RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Identification of the vmaA gene
The only identified V-ATPase subunit in A. nidulans is subunit B. The gene encoding it, vmaB, was identified by its location directly downstream of the tryptophan synthase gene, trpB (Eckert et al., 2000). However, since subunit A is the catalytic subunit, and gene disruptions in both Saccharomyces cerevisiae and N. crassa had been characterized, we wanted to compare the obtained phenotypes and therefore decided to disrupt the corresponding vmaA gene in A. nidulans. The amino acid sequence of subunit A in N. crassa (accession no. J03955) was blasted against the A. nidulans Sequence Database ( http://www.broad.mit.edu/annotation/fungi/aspergillus/index.html ) to obtain the corresponding DNA sequence in A. nidulans. After retrieval of the vmaA candidate gene sequence, it was observed that the predicted two homologous proteins showed a very high similarity, and that, like the Neurospora gene, the Aspergillus gene lacked a canonical transcription initiation site. The A. nidulans vmaA gene is predicted to contain two introns, and has a total length of approximately 2100 bp.

A vmaA disruption phenotype is similar to that caused by bafilomycin or concanamycin
Gene disruptions in A. nidulans can be created by homologous recombination, though high frequencies of non-homologous recombination can be expected to result in random integrations. Thus, the transformants obtained may not contain a disrupted target gene (Fincham, 1989). However, the correct vmaA mutation can be expected to result in a characteristic slow-growing and hyperbranching phenotype. PCR-amplified DNA from a vector plasmid containing the argB marker gene inserted within an internally deleted partial vmaA gene was transformed into an argB deletion strain (Fig. 1; see Methods). Two out of the approximately 200 arginine-prototrophic transformants showed the expected phenotype. One of these was later discarded since Southern blot analysis failed to confirm the correct mutation (data not shown). The other mutant strain (PM6) was crossed with a strain that completely lacked the argB gene to ascertain that the obtained phenotype was due to vmaA disruption. Cleistothecia were collected, separated from conidia and vegetative mycelia, and ascospores (all wild-type-like) were released. When selective medium was used, all viable ascospores showed the expected vmaA1 mutant phenotype (here exemplified by PM7). Fig. 2 shows a Southern blot analysis confirming integration into the vmaA gene. An interesting observation is that the band position is different for all strains. The additional variation between the two mutants indicates an introduced mutation in the PstI site. Alternatively, the vmaA allele, in either or both mutant strains, has been rearranged upon recombination.



View larger version (90K):
[in this window]
[in a new window]
 
Fig. 2. Southern blot analysis of vma1 and wild-type genomic DNA. PstI-digested genomic DNA was subjected to Southern blot analysis to test for disruption of the vmaA gene. DNA was from the following strains: Lane 1, A851; lane 2, PW1; lane 3, PM6; lane 4, PM7. Lane 5, control 6·6 kb.

 
Phenotypic characterization of the vmaA1 mutant strain
The main characteristic of the mutant strain is an extremely low growth rate. After inoculation on minimal medium at 30 °C, germlings from single ascospores appeared after 2 days. After 3 days, the mean colony diameter was 2 mm (wild-type colonies normally grow to 40 mm after 3 days). Another general characteristic is the hyperbranching phenotype with swollen hyphae, which could be observed both in the vmaA mutant strain and after treatment with bafilomycin or concanamycin. In both situations the morphology showed some variations, i.e. the morphology was more severely affected in some hyphal tips than in others (Fig. 3). The most dramatic changes were obtained when the fungus was treated with at least 40 µg bafilomycin B1 ml-1 or 20 µg concanamycin A ml-1. Upon exposure of the mutant strain to higher concentrations of the antibiotics, no additional change in growth and morphology was observed. Thus, this phenotype strongly suggests that the V-ATPase is the only target also in A. nidulans; the severely inhibited growth and altered morphology caused by the antibiotics is consistent with inhibition of the V-ATPase. Even after prolonged incubation (2 weeks) the diameter of the vmaA1 mutant strains did not increase beyond 1 cm (PM6) or 2 cm (PM7). In addition, the mutant was unable to form differentiated hyphae and produce spores. In contrast, the mutant strain formed large vacuoles in older mycelia that were indistinguishable from wild-type (data not shown). A slower growth rate after prolonged incubation might have been due to lack of essential nutrients or evaporation of water on the plate. The slightly higher growth rate of strain PM7 is probably due to methionine prototrophy. In submerged cultures, the ascospores did germinate but the growth rate was even lower than on solid media. Therefore, DNA extraction was carried out from mycelia that had been collected from cellophane-covered agar plates. In cultures grown in liquid medium on a shaker (100 r.p.m.) growth of the fungus was arrested. This close-to-lethality of the vma1 mutation under normal conditions rendered further studies difficult. Establishment of growth conditions able to suppress or compensate for the mutation would thus be desirable. In S. cerevisiae, it has been reported that the growth rate of cells carrying a mutated V-ATPase subunit can be restored by adding 5 mM Fe2+ or Fe3+ (Eide et al., 1993). When tested in A. nidulans, the addition of concentrations up to 5 mM of either Fe2+ or Fe3+ failed to restore normal growth rate of the vmaA1 mutants but instead, at the highest concentrations, caused a further reduction in the growth rate; the same effect was seen in the wild-type strain. In addition, the number of germinated ascospores was decreased. Furthermore, we could not detect any difference in toxicity between Fe2+ and Fe3+ (Table 2).



View larger version (96K):
[in this window]
[in a new window]
 
Fig. 3. Light microscopic images of the mycelial phenotype of the vmaA1 mutant strain, and that of wild-type A. nidulans, untreated and after bafilomycin treatment. (a) Wild-type A. nidulans (48 h incubation); (b) vmaA1 (48 h); (c) wild-type A. nidulans (48 h) treated with bafilomycin B1 (50 µg ml-1); (d) vmaA1 (48 h); (e) Wild-type A. nidulans (48 h) treated with bafilomycin B1 (50 µg ml-1); (f) vmaA1 (48 h) treated with bafilomycin B1 (50 µg ml-1). Bars, 20 µm.

 

View this table:
[in this window]
[in a new window]
 
Table 2. Growth of the vmaA1 mutant strain in minimal medium at different pH values and concentrations of metal ions

+++, No change in growth rate compared to standard conditions; ++, reduced growth; +, germinated ascospores/conidia, but extremely slow growth; -, lethality observed under these conditions.

 
Alkaline pH is lethal for the vmaA1 strain
V-ATPases keep vacuoles acidified by pumping protons from the cytoplasm into the vacuole. In both N. crassa and S. cerevisisae, V-ATPase mutants fail to grow at alkaline pH (Bachhawat et al., 1993; Bowman et al., 2000). To test whether this also holds true for A. nidulans, we inoculated ascospores from the PM6xA851 cross on plates at five different pH values (4·9, 5·8, 6·5, 7·5 and 8·5). The mutant strain was non-viable at pH 7·5 and 8·5, whereas no significant growth-rate effects were detected at the three lowest pH values (Table 2). The same lethality was observed irrespective of whether parts of vmaA1 mutant mycelium, or ascospores, were transferred to the plates. These results indicate that, in agreement with reports from both S. cerevisiae and N. crassa, the A. nidulans V-ATPase subunit A is essential for the fungus at alkaline pH, probably because of the additional activity required to maintain an acidified vacuole interior.

Sensitivity to zinc is increased in the vmaA1 mutant
By mRNA differential display, we previously identified a gene that displayed 20-fold downregulated expression upon treatment with bafilomycin B1. This gene has previously been described as aspnd1 and is a zinc-binding protein involved in zinc uptake (Segurado et al., 1999). Since high zinc concentrations have been reported to be toxic for V-ATPase mutants in N. crassa and S. cerevisiae (Bachhawat et al., 1993; Bowman et al., 2000; Eide et al., 1993; Ramsay & Gadd, 1997), the same toxicity could be expected for the vmaA1 mutant in A. nidulans. This is because a downregulated expression of aspnd1 in the fungus might reduce the uptake of toxic zinc ions. We tested this conjecture by asking whether vmaA1 mutant ascospores were able to germinate in the presence of varying concentrations of Zn2+ (see Methods). In Aspergillus minimal medium, the Zn2+ concentration is approximately 0·15 mM; this was increased to 0·5, 2·0 or 5·0 mM for the tests. At the highest Zn2+ concentration tested, growth of the mutant strain was prevented. At 2·0 mM Zn2+ a substantial decrease in both growth rate and the number of germinated spores was observed, whereas 0·5 mM Zn2+ had no effect. As with increased pH, the lethality caused by zinc was identical for mycelia and ascospores. The wild-type strain was only moderately affected with respect to growth rate and sporulation even at the highest Zn2+ concentration, indicating that an active V-ATPase can confer protection against elevated Zn2+concentrations (Table 2).

Conclusions
We have shown that an intact subunit A of the V-ATPase is essential for normal growth and morphology in A. nidulans. The vmaA1 mutation is lethal at alkaline pH and at high levels of Zn2+. Treatment of A. nidulans with high concentrations of bafilomycin or concanamycin results in a similar phenotype. This indicates that the V-ATPase is the main, or even only, target for the antibiotics. Most of these results are in line with earlier findings in S. cerevisiae and N. crassa, suggesting that the functions of the V-ATPase are the same in all ascomycetes. Although most features of the A. nidulans V-ATPase mutant strain resemble those of the N. crassa mutant, there is a noteworthy difference concerning the growth and viability of the mutants. In A. nidulans, the mutant strain showed an extremely reduced growth, increased branching and more extensively swollen mycelia at acidic pH, and failed to grow altogether under alkaline conditions. By contrast, the N. crassa vmaA mutant is viable at acidic pH and covers a Petri dish within 4 days (Bowman et al., 2000). Wild-type N. crassa is characterized by higher growth rate and different growth optima than A. nidulans. Thus, it is likely that the variations in the subunit A mutants can be attributed to general differences in physiology between the two filamentous fungi, rather than to the specific function of the V-ATPase.


   ACKNOWLEDGEMENTS
 
This work was supported by the Swedish Council for Forestry and Agricultural Research (SJFR/FORMAS).


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Bachhawat, A. K., Manolson, M. F., Murdock, D. G., Garman, J. D. & Jones, E. W. (1993). The Vph2-gene encodes a 25 kDa protein required for activity of the yeast vacuolar H+-ATPase. Yeast 9, 175–184.[Medline]

Bowman, E. J., Siebers, A. & Altendorf, K. (1988). Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells. Proc Natl Acad Sci U S A 85, 7972–7976.[Abstract]

Bowman, E. J., Kendle, R. & Bowman, B. J. (2000). Disruption of vma-1, the gene encoding the catalytic subunit of the vacuolar H+-ATPase, causes severe morphological changes in Neurospora crassa. J Biol Chem 275, 167–176.[Abstract/Free Full Text]

Dröse, S., Bindseil, K. U., Bowman, E. J., Siebers, A., Zeeck, A. & Altendorf, K. (1993). Inhibitory effect of modified bafilomycins and concanamycins on P- and V-type adenosinetriphosphatases. Biochemistry 32, 3902–3906.[Medline]

Eckert, S. E., Kubler, E., Hoffmann, B. & Braus, G. H. (2000). The tryptophan synthase-encoding trpB gene of Aspergillus nidulans is regulated by the cross-pathway control system. Mol Gen Genet 263, 867–876.[CrossRef][Medline]

Eide, D. J., Bridgham, J. T., Zhao, Z. & Mattoon, J. R. (1993). The vacuolar H+-ATPase of Saccharomyces cerevisiae is required for efficient copper detoxification, mitochondrial function, and iron metabolism. Mol Gen Genet 241, 447–456.[Medline]

Fincham, J. R. (1989). Transformation in fungi. Microbiol Rev 53, 148–170.[Medline]

Forgac, M. (1989). Structure and function of vacuolar class of ATP-driven proton pumps. Physiol Rev 69, 765–796.[Free Full Text]

Frändberg, E. & Schnürer, J. (1998). Antifungal activity of chitinolytic bacteria isolated from airtight stored cereal grain. Can J Microbiol 44, 121–127.[CrossRef]

Frändberg, E., Petersson, C., Lundgren, L. N. & Schnürer, J. (2000). Streptomyces halstedii K122 produces the antifungal compounds bafilomycin B1 and C1. Can J Microbiol 46, 753–758.[CrossRef][Medline]

Kaminskyj, S. G. W. (2001). Fundamentals of growth, storage, genetics and microscopy of Aspergillus nidulans. Fungal Genet Newsl 48, 25–31.

Klionsky, D. J., Herman, P. K. & Emr, S. D. (1990). The fungal vacuole: composition, function, and biogenesis. Microbiol Rev 54, 266–292.[Medline]

Melin, P., Schnürer, J. & Wagner, E. G. H. (1999). Changes in Aspergillus nidulans gene expression induced by bafilomycin, a Streptomyces-produced antibiotic. Microbiology 145, 1115–1122.[Abstract]

Melin, P., Schnürer, J. & Wagner, E. G. H. (2002). Proteome analysis of Aspergillus nidulans reveals proteins associated with the response to the antibiotic concanamycin A, produced by Streptomyces species. Mol Genet Genom 267, 695–702.[CrossRef][Medline]

Melin, P., Schnürer, J. & Wagner, E. G. H. (2003). Characterization of phiA, a gene essential for phialide development in Aspergillus nidulans. Fungal Genet Biol 40, 234–241.[CrossRef][Medline]

Pontecorvo, G., Roper, J. A. L. M. H., MacDonald, K. D. & Bufton, A. W. J. (1953). The genetics of Aspergillus nidulans. Adv Genet 5, 141–237.

Ramsay, L. M. & Gadd, G. M. (1997). Mutants of Saccharomyces cerevisiae defective in vacuolar function confirm a role for the vacuole in toxic metal ion detoxification. FEMS Microbiol Let 152, 293–298.[CrossRef][Medline]

Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Segurado, M., Lopez-Aragon, R., Calera, J. A., Fernandez-Abalos, J. M. & Leal, F. (1999). Zinc-regulated biosynthesis of immunodominant antigens from Aspergillus spp. Infect Immun 67, 2377–2382.[Abstract/Free Full Text]

Talbot, N. J. (2001). Nucleic acid isolation and analysis. In Molecular and Cellular Biology of Filamentous Fungi, pp. 23–26. Edited by N. J. Talbot. Oxford: Oxford University Press.

Weber, R. W. S. (2002). Vacuoles and the fungal lifestyle. Mycologist 16, 10–20.[CrossRef]

Werner, G. & Hagenmaier, H. (1984). Metabolic products of microorganisms. 224. Bafilomycins, a new group of macrolide antibiotics: production, isolation, chemical structure and biological activity. J Antibiot 37, 110–117.[Medline]

Received 1 October 2003; revised 27 November 2003; accepted 2 December 2003.



This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Melin, P.
Articles by Wagner, E. G. H.
Articles citing this Article
PubMed
PubMed Citation
Articles by Melin, P.
Articles by Wagner, E. G. H.
Agricola
Articles by Melin, P.
Articles by Wagner, E. G. H.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
INT J SYST EVOL MICROBIOL MICROBIOLOGY J GEN VIROL
J MED MICROBIOL ALL SGM JOURNALS
Copyright © 2004 Society for General Microbiology.