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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
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concentration results in increased swelling of Aspergillus mycelium, and growth is almost completely inhibited (unpublished observations).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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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
).
|
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
|
|
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 |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
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, 79727976.[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, 167176.
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, 39023906.[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, 867876.[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, 447456.[Medline]
Fincham, J. R. (1989). Transformation in fungi. Microbiol Rev 53, 148170.[Medline]
Forgac, M. (1989). Structure and function of vacuolar class of ATP-driven proton pumps. Physiol Rev 69, 765796.
Frändberg, E. & Schnürer, J. (1998). Antifungal activity of chitinolytic bacteria isolated from airtight stored cereal grain. Can J Microbiol 44, 121127.[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, 753758.[CrossRef][Medline]
Kaminskyj, S. G. W. (2001). Fundamentals of growth, storage, genetics and microscopy of Aspergillus nidulans. Fungal Genet Newsl 48, 2531.
Klionsky, D. J., Herman, P. K. & Emr, S. D. (1990). The fungal vacuole: composition, function, and biogenesis. Microbiol Rev 54, 266292.[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, 11151122.[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, 695702.[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, 234241.[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, 141237.
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, 293298.[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, 23772382.
Talbot, N. J. (2001). Nucleic acid isolation and analysis. In Molecular and Cellular Biology of Filamentous Fungi, pp. 2326. Edited by N. J. Talbot. Oxford: Oxford University Press.
Weber, R. W. S. (2002). Vacuoles and the fungal lifestyle. Mycologist 16, 1020.[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, 110117.[Medline]
Received 1 October 2003;
revised 27 November 2003;
accepted 2 December 2003.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |