- Email: [email protected]
Identification and linkage mapping of the phsA gene of Aspergillus nidulans, where mutation affects growth and pigmentation of colonies in a temperature- and pH-dependent way
FEMS Microbiology Letters 171 (1999) 103^106
Identi¢cation and linkage mapping of the phsA gene of Aspergillus nidulans, where mutation a¡ects growth and pigmentation of colonies in a temperature- and pH-dependent way Sara C. Cuadros a , Nilce M. Martinez-Rossi a , Antonio Rossi b; * b
a Departamento de Geneètica, Faculdade de Medicina de Ribeiraìo Preto, USP, 14049-900 Ribeiraìo Preto, SP, Brazil Departamento de Qu|èmica, Faculdade de Filoso¢a, Cieências e Letras de Ribeiraìo Preto, USP, 14040-901 Ribeiraìo Preto, SP, Brazil
Received 12 November 1998; received in revised form 14 December 1998; accepted 14 December 1998
Abstract We report the isolation and characterization of a mutant strain of the mold Aspergillus nidulans showing an altered response to environmental pH, including a reduction in its pH range for growth and the production of a melanin-like pigment at alkaline pH. We also show that the mutant strain is not detergent-sensitive and that its acid sensitivity is osmotically remediable with 0.5 M NaCl or 1.0 M sorbitol. Furthermore, the mutant phenotype is temperature-remediable with respect to pigmentation, extent of conidiation and growth diameter, with the restoration of a wild-type phenotype to the mutant strain being observed at 28³C. On the other hand, the severity of the mutant phenotype is increased at 40³C. Genetic analysis shows that this pH- and temperature-sensitive mutation, named phsA1, is located on the right arm of linkage group I of A. nidulans, between pabaA and yA, and that mutation phsA1 is recessive. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Aspergillus nidulans; pH sensitivity; Melanin-like pigment
1. Introduction Aspergillus nidulans, an ascomycete fungus capable of growing over a wide pH range [1], has been extensively used in genetic and biochemical approaches over the last 50 years, being thus well characterized and very appropriate as an experimental model [2]. This interesting physiological versatility concerning the response to ambient pH implies the existence of mechanisms that regulate not only the homeostatic * Corresponding author. Tel.: +55 (16) 6023749; Fax: +55 (16) 6338151; E-mail: [email protected]
pH but also the expression of structural genes for permeases and extracellular enzymes, ensuring that these enzymes will be secreted only at pH values at which they can function e¡ectively [3^5]. However, in spite of the extensive work done to investigate the pH signal transduction pathway and pH-regulated genes involved in the synthesis of these proteins and secondary metabolites, much about the pH regulatory system of A. nidulans remains to be clari¢ed [6^10]. In order to investigate other physiological responses possibly involved in the adaptation and survival under extremes pH conditions, our ¢rst aim
0378-1097 / 99 / $19.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 5 8 7 - 4
FEMSLE 8581 5-2-99
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S.C. Cuadros et al. / FEMS Microbiology Letters 171 (1999) 103^106
2. Materials and methods
diate plating of these spores onto complete medium, pH 6.5. We selected 100 colonies at random and analyzed the pH response of these strains. pH-sensitive mutants were identi¢ed as showing poor growth on complete medium bu¡ered at pH 3.0 and pH 8.5 compared to the wild-type strain pattern of growth.
2.1. Growth media and strains
2.3. Characterization of a phs strain
The growth media used were previously described by Cove [11]. All media contained 55 mM glucose as the carbon source and 70 mM sodium nitrate as the nitrogen source (¢nal concentrations). Citric acid (50 mM), piperazine-N,NP-bis-2-ethanesulfonic acid (PIPES) (50 mM) and Tris-hydrochloride (50 mM) were added to solid complete medium as appropriate bu¡ers for each pH range studied. NaCl (0.5 M) or sorbitol (1.0 M) were used as osmotic stabilizer. Minimal medium was used for auxotrophy analysis and adequate supplements were added at the time for plating. The incubation temperature was 37³C and incubation time was 48 h. Strain pabaA1 (p-aminobenzoate requiring) and the multiple marker master strains E and F (MSE and MSF are the FGSC strains A288 and A283, respectively) of A. nidulans were used.
Among the various strains selected, one showing a reduced pH range for growth and altered pigmentation at alkaline pH was chosen at random for further study. We analyzed the growth of this strain on solid complete medium at di¡erent osmolarity by adding NaCl to the medium. Growth was estimated by determining the colony diameter of 20 colonies inoculated in groups of four in 90 mm diameter dishes. We further investigated growth on solid complete medium at di¡erent temperatures (28³C, 37³C and 40³C) and in the presence of sodium dodecylsulfate (SDS), an ionic detergent, at the following ¢nal concentrations: 34 WM, 173 WM, 260 WM, and 695 WM.
was to isolate and characterize mutants of A. nidulans showing an altered pH sensitivity for growth and then locate these mutations.
2.2. Mutagenesis and selection of mutants pH-sensitive mutations (designated phs) were selected after UV mutagenesis in a pabaA1 strain of A. nidulans, until 95% kill was achieved, and imme-
2.4. Genetic techniques Standard A. nidulans genetic techniques were those of Pontecorvo et al. [12]. Diploids were obtained using the technique described by Roper [13]. Allocation of mutations to their linkage groups using the parasexual cycle, as described by McCully and Forbes [14], was facilitated by the use of benlate [15]. Haploidization was performed on complete medium
Table 1 E¡ect of culture conditions on colony radial growtha of strains pabaA1 and pabaA1 phsA1 of A. nidulans on solid complete medium, 37³C Strain
Incubation time (h)
pH and e¡ect of increased osmolarity pH 3.0 ^
pabaA1 phsA1
pabaA1
a
26 50 72 26 50 72
63.5 þ 5.6
pH 5.0
pH 7.0
pH 9.0
0.5 M NaCl
^
0.5 M NaCl
^
0.5 M NaCl
100.8 þ 5.1
130.3 þ 4.1
121.0 þ 8.1
126.5 þ 6.1
113.3 þ 5.7
136.3 þ 9.0 225.5 þ 8.1 240.8 þ 11.7 425.5 þ 9.4 78.2 þ 3.3 100.5 þ 7.4 195.3 þ 6.2 226.8 þ 4.4 368.8 þ 7.9 429.5 þ 10.0
270.5 þ 8.9 289.0 þ 9.1 454.2 þ 17.7 519.0 þ 18.3 132.8 þ 4.1 131.8 þ 3.7 284.0 þ 7.6 295.0 þ 10.5 502.0 þ 11.0 523.5 þ 15.0
278.0 þ 12.4 235.5 þ 10.0 509.5 þ 11.9 456.0 þ 12.3 132.5 þ 3.8 116.8 þ 4.1 296.0 þ 4.9 251.0 þ 7.9 523.6 þ 7.4 445.9 þ 16.2
^
0.5 M NaCl 49.0 þ 3.1
54.5 þ 5.8
112.5 þ 5.5 116.8 þ 6.6 211.5 þ 9.3 231.0 þ 10.7 76.8 þ 4.1 68.8 þ 5.6 150.0 þ 10.6 171.3 þ 3.6 327.5 þ 7.9 262.5 þ 11.2
Average diameter (mm) of 20 colonies þ S.D. Two independent experiments were conducted and representative results are shown.
FEMSLE 8581 5-2-99
S.C. Cuadros et al. / FEMS Microbiology Letters 171 (1999) 103^106
at 37³C, on which both master strains E and F and strain phsA1 showed almost the same sensitivity to benlate.
3. Results and discussion We isolated a mutant strain, named phsA1, showing an increased sensitivity to both acid and alkaline pH, characterized by reduced growth and altered morphology at extreme pH values when compared to the wild-type strain (Table 1). We also observed an increase in pigmentation and a reduction in conidiation of the colonies as the growth pH increased from 6.0 to 8.5. This mutant phenotype was temperature-remediable with respect to pigmentation, extent of conidiation and growth diameter, because restoration of a wild-type phenotype to the mutant strain was observed at 28³C. On the other hand, the severity of the mutant phenotype was increased at 40³C. Also, growth of the phsA1 strain on media supplemented with di¡erent concentrations of SDS, pH 6.8, showed no altered response when compared to the wild-type strain. However, acid sensitivity was osmotically remediable with 0.5 M NaCl (Table 1), leading to the restoration of the wild-type phenotype with respect to colony diameter and morphology. A preliminary experiment indicated that 1 M sorbitol resembles 0.5 M NaCl in achieving remediability of the acid sensitivity of strain phsA1. The alkaline sensitivity phenotype, however, was not osmotically remediable. Taken together, these results indicate that mutation phsA1 probably promoted alterations in the structural organization of the cell (plasma membrane, cell wall, etc.), leading to its pH sensitivity. We isolated diploids between the phsA1 strain and the master strains E and F which were not pH-sensitive and had no increased pigmentation, showing that these characters are recessive. The parasexual analysis of 30 segregants from the diploid with MSE assigned both the pH sensitivity and the pigmentation phenotypes to linkage group I. Analysis of 240 segregants from a cross to MSE showed that these phenotypes are probably related to a single gene. On the basis of recombination rates, mutation phsA1 was mapped on the right arm of chromosome I. Our results also showed it to lie between pabaA1 and yA2, 9.1 cM from pabaA and 17.7 cM from yA.
105
However, no systematic e¡ort to precisely locate the phsA gene with respect to other genes located between the pabaA and yA genes was made. pH-conditional defects in growth and morphogenesis which are evident at alkaline pH but absent at acidic pH have been reported for Candida albicans and shown to be related to virulence in this opportunistic fungal pathogen [16]. Some environmentally regulated genes a¡ecting growth have been described in the genus Aspergillus [17,18]. Recent work has suggested that the alb1 gene of A. fumigatus, which is involved in melanin biosynthesis, has a role in the modulation of virulence [19]. We believe that further characterization of strain phsA1 will clear up the role of this temperature- and pH-regulated response gene that is also involved in production of a melanin-like pigment in A. nidulans.
Acknowledgments We thank CNPq and FAPESP for ¢nancial support.
References [1] Rossi, A. and Arst, H.N. Jr. (1990) Mutants of Aspergillus nidulans able to grow at extremely acidic pH acidify the medium less than wild-type when grown at more moderate pH. FEMS Microbiol. Lett. 66, 51^54. [2] Martinelli, S.D. and Kinghorn, J.R. (Eds.) (1994) Aspergillus: 50 Years On. Elsevier, Amsterdam. [3] Nahas, E., Terenzi, H.F. and Rossi, A. (1982) E¡ect of carbon source and pH on the production and secretion of acid phosphatase (EC 3.1.3.2) and alkaline phosphatase (EC 3.1.3.1) in Neurospora crassa. J. Gen. Microbiol. 128, 2017^2021. [4] Caddick, M.X., Brownlee, A.G. and Arst, H.N. Jr. (1986) Regulation of gene expression by pH of the growth medium in Aspergillus nidulans. Mol. Gen. Genet. 203, 346^353. [5] Maccheroni, W. Jr., Pombeiro, S.R.C., Martinez-Rossi, N.M. and Rossi, A. (1991) pH and acid phosphatase determination after growth of Aspergillus nidulans on solid medium. Fungal Genet. Newslett. 38, 78. [6] Espeso, E.A., Tilburn, J., Arst, H.N. Jr. and Penìalva, M.A. (1993) pH regulation is a major determinant in expression of a fungal penicillin biosynthetic gene. EMBO J. 12, 3947^3956. [7] Katz, M.E., Flynn, P.K., vanKuyk, P.A. and Cheetham, B.F. (1996) Mutations a¡ecting extracellular protease production in the ¢lamentous fungus Aspergillus nidulans. Mol. Gen. Genet. 250, 715^724. [8] Tilburn, J., Sarkar, S., Widdick, D.A., Espeso, E.A., Orejas,
FEMSLE 8581 5-2-99
106
[9]
[10]
[11]
[12]
[13] [14]
S.C. Cuadros et al. / FEMS Microbiology Letters 171 (1999) 103^106 M., Mungroo, J., Penìalva, M.A. and Arst, H.N. Jr. (1995) The Aspergillus PacC zinc ¢nger transcription factor mediates regulation of both acid and alkaline expressed genes by ambient pH. EMBO J. 14, 779^790. Orejas, M., Espeso, E.A., Tilburn, J., Sarkar, S., Arst, H.N. Jr. and Penìalva, M.A. (1995) Activation of the Aspergillus PacC transcription factor in response to alkaline ambient pH requires proteolysis of the carboxy-terminal moiety. Genes Dev. 9, 1622^1632. Bergh, K.T. and Brakhage, A.A. (1998) Regulation of the Aspergillus nidulans penicillin biosynthesis gene acvA (pcbAB) by amino acids: Implication for the involvement of transcription factor PacC. Appl. Environ. Microbiol. 64, 843^849. Cove, D.J. (1966) The induction and repression of nitrate reductase in the fungus Aspergillus nidulans. Biochim. Biophys. Acta 113, 51^56. Pontecorvo, G., Roper, J.A., Hemmons, L.M., MacDonald, K.D. and Bufton, A.W.J. (1953) The genetics of Aspergillus nidulans. Adv. Genet. 5, 141^238. Roper, J.A. (1952) Production of heterozygous diploids in ¢lamentous fungi. Experientia 8, 14^15. McCully, K.S. and Forbes, E. (1965) The use p-£uorophenyl-
[15] [16]
[17]
[18]
[19]
alanine with master strains of Aspergillus nidulans for assigning genes to linkage groups. Genet. Res. 6, 352^259. Hastie, A.C. (1970) Benlate induced instability of Aspergillus diploids. Nature 226, 771. Muhlschlegel, F.A. and Fonzi, W.A. (1997) PHR2 of Candida albicans encodes a functional homolog of the pH-regulated gene PHR1 with an inverted pattern of pH-dependent expression. Mol. Cell. Biol. 17, 5960^5967. Hensel, M., Arst, H.N. Jr., Aufauvre-Brown, A. and Holden, D.W. (1998) The role of the Aspergillus fumigatus areA gene in invasive pulmonary aspergillosis. Mol. Gen. Genet. 258, 553^557. Jahn, B., Koch, A., Schmidt, A., Wanner, G., Gehringer, H., Bhakdi, S. and Brakhage, A.A. (1997) Isolation and characterization of a pigmentless-conidium mutant of Aspergillus fumigatus with altered conidial surface and reduced virulence. Infect. Immun. 65, 5110^5117. Tsai, H.F., Chang, Y.C., Washburn, R.G., Wheeler, M.H. and Kwon, K.J. (1998) The developmentally regulated alb1 gene of Aspergillus fumigatus: its role in modulation of conidial morphology and virulence. J. Bacteriol. 180, 3031^ 3038.
FEMSLE 8581 5-2-99
Identi¢cation and linkage mapping of the phsA gene of Aspergillus nidulans, where mutation a¡ects growth and pigmentation of colonies in a temperature- and pH-dependent way Sara C. Cuadros a , Nilce M. Martinez-Rossi a , Antonio Rossi b; * b
a Departamento de Geneètica, Faculdade de Medicina de Ribeiraìo Preto, USP, 14049-900 Ribeiraìo Preto, SP, Brazil Departamento de Qu|èmica, Faculdade de Filoso¢a, Cieências e Letras de Ribeiraìo Preto, USP, 14040-901 Ribeiraìo Preto, SP, Brazil
Received 12 November 1998; received in revised form 14 December 1998; accepted 14 December 1998
Abstract We report the isolation and characterization of a mutant strain of the mold Aspergillus nidulans showing an altered response to environmental pH, including a reduction in its pH range for growth and the production of a melanin-like pigment at alkaline pH. We also show that the mutant strain is not detergent-sensitive and that its acid sensitivity is osmotically remediable with 0.5 M NaCl or 1.0 M sorbitol. Furthermore, the mutant phenotype is temperature-remediable with respect to pigmentation, extent of conidiation and growth diameter, with the restoration of a wild-type phenotype to the mutant strain being observed at 28³C. On the other hand, the severity of the mutant phenotype is increased at 40³C. Genetic analysis shows that this pH- and temperature-sensitive mutation, named phsA1, is located on the right arm of linkage group I of A. nidulans, between pabaA and yA, and that mutation phsA1 is recessive. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Aspergillus nidulans; pH sensitivity; Melanin-like pigment
1. Introduction Aspergillus nidulans, an ascomycete fungus capable of growing over a wide pH range [1], has been extensively used in genetic and biochemical approaches over the last 50 years, being thus well characterized and very appropriate as an experimental model [2]. This interesting physiological versatility concerning the response to ambient pH implies the existence of mechanisms that regulate not only the homeostatic * Corresponding author. Tel.: +55 (16) 6023749; Fax: +55 (16) 6338151; E-mail: [email protected]
pH but also the expression of structural genes for permeases and extracellular enzymes, ensuring that these enzymes will be secreted only at pH values at which they can function e¡ectively [3^5]. However, in spite of the extensive work done to investigate the pH signal transduction pathway and pH-regulated genes involved in the synthesis of these proteins and secondary metabolites, much about the pH regulatory system of A. nidulans remains to be clari¢ed [6^10]. In order to investigate other physiological responses possibly involved in the adaptation and survival under extremes pH conditions, our ¢rst aim
0378-1097 / 99 / $19.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 5 8 7 - 4
FEMSLE 8581 5-2-99
104
S.C. Cuadros et al. / FEMS Microbiology Letters 171 (1999) 103^106
2. Materials and methods
diate plating of these spores onto complete medium, pH 6.5. We selected 100 colonies at random and analyzed the pH response of these strains. pH-sensitive mutants were identi¢ed as showing poor growth on complete medium bu¡ered at pH 3.0 and pH 8.5 compared to the wild-type strain pattern of growth.
2.1. Growth media and strains
2.3. Characterization of a phs strain
The growth media used were previously described by Cove [11]. All media contained 55 mM glucose as the carbon source and 70 mM sodium nitrate as the nitrogen source (¢nal concentrations). Citric acid (50 mM), piperazine-N,NP-bis-2-ethanesulfonic acid (PIPES) (50 mM) and Tris-hydrochloride (50 mM) were added to solid complete medium as appropriate bu¡ers for each pH range studied. NaCl (0.5 M) or sorbitol (1.0 M) were used as osmotic stabilizer. Minimal medium was used for auxotrophy analysis and adequate supplements were added at the time for plating. The incubation temperature was 37³C and incubation time was 48 h. Strain pabaA1 (p-aminobenzoate requiring) and the multiple marker master strains E and F (MSE and MSF are the FGSC strains A288 and A283, respectively) of A. nidulans were used.
Among the various strains selected, one showing a reduced pH range for growth and altered pigmentation at alkaline pH was chosen at random for further study. We analyzed the growth of this strain on solid complete medium at di¡erent osmolarity by adding NaCl to the medium. Growth was estimated by determining the colony diameter of 20 colonies inoculated in groups of four in 90 mm diameter dishes. We further investigated growth on solid complete medium at di¡erent temperatures (28³C, 37³C and 40³C) and in the presence of sodium dodecylsulfate (SDS), an ionic detergent, at the following ¢nal concentrations: 34 WM, 173 WM, 260 WM, and 695 WM.
was to isolate and characterize mutants of A. nidulans showing an altered pH sensitivity for growth and then locate these mutations.
2.2. Mutagenesis and selection of mutants pH-sensitive mutations (designated phs) were selected after UV mutagenesis in a pabaA1 strain of A. nidulans, until 95% kill was achieved, and imme-
2.4. Genetic techniques Standard A. nidulans genetic techniques were those of Pontecorvo et al. [12]. Diploids were obtained using the technique described by Roper [13]. Allocation of mutations to their linkage groups using the parasexual cycle, as described by McCully and Forbes [14], was facilitated by the use of benlate [15]. Haploidization was performed on complete medium
Table 1 E¡ect of culture conditions on colony radial growtha of strains pabaA1 and pabaA1 phsA1 of A. nidulans on solid complete medium, 37³C Strain
Incubation time (h)
pH and e¡ect of increased osmolarity pH 3.0 ^
pabaA1 phsA1
pabaA1
a
26 50 72 26 50 72
63.5 þ 5.6
pH 5.0
pH 7.0
pH 9.0
0.5 M NaCl
^
0.5 M NaCl
^
0.5 M NaCl
100.8 þ 5.1
130.3 þ 4.1
121.0 þ 8.1
126.5 þ 6.1
113.3 þ 5.7
136.3 þ 9.0 225.5 þ 8.1 240.8 þ 11.7 425.5 þ 9.4 78.2 þ 3.3 100.5 þ 7.4 195.3 þ 6.2 226.8 þ 4.4 368.8 þ 7.9 429.5 þ 10.0
270.5 þ 8.9 289.0 þ 9.1 454.2 þ 17.7 519.0 þ 18.3 132.8 þ 4.1 131.8 þ 3.7 284.0 þ 7.6 295.0 þ 10.5 502.0 þ 11.0 523.5 þ 15.0
278.0 þ 12.4 235.5 þ 10.0 509.5 þ 11.9 456.0 þ 12.3 132.5 þ 3.8 116.8 þ 4.1 296.0 þ 4.9 251.0 þ 7.9 523.6 þ 7.4 445.9 þ 16.2
^
0.5 M NaCl 49.0 þ 3.1
54.5 þ 5.8
112.5 þ 5.5 116.8 þ 6.6 211.5 þ 9.3 231.0 þ 10.7 76.8 þ 4.1 68.8 þ 5.6 150.0 þ 10.6 171.3 þ 3.6 327.5 þ 7.9 262.5 þ 11.2
Average diameter (mm) of 20 colonies þ S.D. Two independent experiments were conducted and representative results are shown.
FEMSLE 8581 5-2-99
S.C. Cuadros et al. / FEMS Microbiology Letters 171 (1999) 103^106
at 37³C, on which both master strains E and F and strain phsA1 showed almost the same sensitivity to benlate.
3. Results and discussion We isolated a mutant strain, named phsA1, showing an increased sensitivity to both acid and alkaline pH, characterized by reduced growth and altered morphology at extreme pH values when compared to the wild-type strain (Table 1). We also observed an increase in pigmentation and a reduction in conidiation of the colonies as the growth pH increased from 6.0 to 8.5. This mutant phenotype was temperature-remediable with respect to pigmentation, extent of conidiation and growth diameter, because restoration of a wild-type phenotype to the mutant strain was observed at 28³C. On the other hand, the severity of the mutant phenotype was increased at 40³C. Also, growth of the phsA1 strain on media supplemented with di¡erent concentrations of SDS, pH 6.8, showed no altered response when compared to the wild-type strain. However, acid sensitivity was osmotically remediable with 0.5 M NaCl (Table 1), leading to the restoration of the wild-type phenotype with respect to colony diameter and morphology. A preliminary experiment indicated that 1 M sorbitol resembles 0.5 M NaCl in achieving remediability of the acid sensitivity of strain phsA1. The alkaline sensitivity phenotype, however, was not osmotically remediable. Taken together, these results indicate that mutation phsA1 probably promoted alterations in the structural organization of the cell (plasma membrane, cell wall, etc.), leading to its pH sensitivity. We isolated diploids between the phsA1 strain and the master strains E and F which were not pH-sensitive and had no increased pigmentation, showing that these characters are recessive. The parasexual analysis of 30 segregants from the diploid with MSE assigned both the pH sensitivity and the pigmentation phenotypes to linkage group I. Analysis of 240 segregants from a cross to MSE showed that these phenotypes are probably related to a single gene. On the basis of recombination rates, mutation phsA1 was mapped on the right arm of chromosome I. Our results also showed it to lie between pabaA1 and yA2, 9.1 cM from pabaA and 17.7 cM from yA.
105
However, no systematic e¡ort to precisely locate the phsA gene with respect to other genes located between the pabaA and yA genes was made. pH-conditional defects in growth and morphogenesis which are evident at alkaline pH but absent at acidic pH have been reported for Candida albicans and shown to be related to virulence in this opportunistic fungal pathogen [16]. Some environmentally regulated genes a¡ecting growth have been described in the genus Aspergillus [17,18]. Recent work has suggested that the alb1 gene of A. fumigatus, which is involved in melanin biosynthesis, has a role in the modulation of virulence [19]. We believe that further characterization of strain phsA1 will clear up the role of this temperature- and pH-regulated response gene that is also involved in production of a melanin-like pigment in A. nidulans.
Acknowledgments We thank CNPq and FAPESP for ¢nancial support.
References [1] Rossi, A. and Arst, H.N. Jr. (1990) Mutants of Aspergillus nidulans able to grow at extremely acidic pH acidify the medium less than wild-type when grown at more moderate pH. FEMS Microbiol. Lett. 66, 51^54. [2] Martinelli, S.D. and Kinghorn, J.R. (Eds.) (1994) Aspergillus: 50 Years On. Elsevier, Amsterdam. [3] Nahas, E., Terenzi, H.F. and Rossi, A. (1982) E¡ect of carbon source and pH on the production and secretion of acid phosphatase (EC 3.1.3.2) and alkaline phosphatase (EC 3.1.3.1) in Neurospora crassa. J. Gen. Microbiol. 128, 2017^2021. [4] Caddick, M.X., Brownlee, A.G. and Arst, H.N. Jr. (1986) Regulation of gene expression by pH of the growth medium in Aspergillus nidulans. Mol. Gen. Genet. 203, 346^353. [5] Maccheroni, W. Jr., Pombeiro, S.R.C., Martinez-Rossi, N.M. and Rossi, A. (1991) pH and acid phosphatase determination after growth of Aspergillus nidulans on solid medium. Fungal Genet. Newslett. 38, 78. [6] Espeso, E.A., Tilburn, J., Arst, H.N. Jr. and Penìalva, M.A. (1993) pH regulation is a major determinant in expression of a fungal penicillin biosynthetic gene. EMBO J. 12, 3947^3956. [7] Katz, M.E., Flynn, P.K., vanKuyk, P.A. and Cheetham, B.F. (1996) Mutations a¡ecting extracellular protease production in the ¢lamentous fungus Aspergillus nidulans. Mol. Gen. Genet. 250, 715^724. [8] Tilburn, J., Sarkar, S., Widdick, D.A., Espeso, E.A., Orejas,
FEMSLE 8581 5-2-99
106
[9]
[10]
[11]
[12]
[13] [14]
S.C. Cuadros et al. / FEMS Microbiology Letters 171 (1999) 103^106 M., Mungroo, J., Penìalva, M.A. and Arst, H.N. Jr. (1995) The Aspergillus PacC zinc ¢nger transcription factor mediates regulation of both acid and alkaline expressed genes by ambient pH. EMBO J. 14, 779^790. Orejas, M., Espeso, E.A., Tilburn, J., Sarkar, S., Arst, H.N. Jr. and Penìalva, M.A. (1995) Activation of the Aspergillus PacC transcription factor in response to alkaline ambient pH requires proteolysis of the carboxy-terminal moiety. Genes Dev. 9, 1622^1632. Bergh, K.T. and Brakhage, A.A. (1998) Regulation of the Aspergillus nidulans penicillin biosynthesis gene acvA (pcbAB) by amino acids: Implication for the involvement of transcription factor PacC. Appl. Environ. Microbiol. 64, 843^849. Cove, D.J. (1966) The induction and repression of nitrate reductase in the fungus Aspergillus nidulans. Biochim. Biophys. Acta 113, 51^56. Pontecorvo, G., Roper, J.A., Hemmons, L.M., MacDonald, K.D. and Bufton, A.W.J. (1953) The genetics of Aspergillus nidulans. Adv. Genet. 5, 141^238. Roper, J.A. (1952) Production of heterozygous diploids in ¢lamentous fungi. Experientia 8, 14^15. McCully, K.S. and Forbes, E. (1965) The use p-£uorophenyl-
[15] [16]
[17]
[18]
[19]
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