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Cloning and sequence analysis of cnaA gene encoding the catalytic subunit of calcineurin from Aspergillus oryzae
FEMS Microbiology Letters 204 (2001) 169^174
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Cloning and sequence analysis of cnaA gene encoding the catalytic subunit of calcineurin from Aspergillus oryzae Praveen Rao Juvvadi, Manabu Arioka, Harushi Nakajima, Katsuhiko Kitamoto * Department of Biotechnology, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-Ku, 113-8657 Tokyo, Japan Received 26 June 2001; received in revised form 12 August 2001; accepted 14 August 2001 First published online 26 September 2001
Abstract Calcineurin has been implicated in ion-homeostasis, stress adaptation in yeast and for hyphal growth in filamentous fungi. Genomic DNA and cDNA encoding the catalytic subunit of calcineurin (cnaA) were isolated from Aspergillus oryzae. The cnaA open reading frame extended to 1727 bp and encoded a putative protein of 514 amino acids. Comparative analysis of the nucleotide sequence of cnaA genomic DNA and cDNA confirmed the presence of three introns and a highly conserved calmodulin binding domain. The deduced amino acid sequence was homologous to calcineurin A from Aspergillus nidulans (92%), Neurospora crassa (84%), human (67%), Saccharomyces cerevisiae (58%) and Schizosaccharomyces pombe (54%). Further, A. oryzae cnaA cDNA complemented S. cerevisiae calcineurin disruptant strain (vcmp1 vcmp2), which was not viable in the presence of high concentrations of NaCl (1.2 M) and at alkaline pH 8.5. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Calcineurin A; Protein phosphatase 2B; Calmodulin-dependent protein phosphatase; Aspergillus oryzae
1. Introduction Calcineurin, the only Ca2 /calmodulin-dependent protein phosphatase (serine/threonine protein phosphatase 2B) recognized as yet in fungi, is a heterodimer comprised of two subunits, viz. K-catalytic subunit A (58^64 kDa) that binds to calmodulin and a L-regulatory subunit B (19 kDa) which is structurally similar to calmodulin. The highly conserved nature and ubiquitous presence of this protein phosphatase throughout the animal and plant kingdom have prompted several investigations into its structure and function [1]. While in mammalian tissues its importance has been implicated in neuronal metabolism, T-lymphocyte proliferation and immune suppression [2], current data in Saccharomyces cerevisiae suggest its
* Corresponding author. Tel. : +81 (3) 5841-5161; Fax: +81 (3) 5841-8033. E-mail address : [email protected] (K. Kitamoto).
requirement for ion-homeostasis, salt stress adaptation, recovery from pheromone-induced growth arrest and in the regulation of alkaline pH-mediated growth [3]. In the basidiomycetous pathogenic fungi Cryptococcus neoformans calcineurin regulates mating, virulence and is required for growth at high temperatures [4]. Among the ¢lamentous fungi its relevance has been reported for hyphal extension in Neurospora crassa, cell cycle regulation in Aspergillus nidulans and secondary metabolism in Aspergillus parasiticus [5^7]. Molecular studies in Aspergillus oryzae, the koji mold, an important fungus widely used in Japanese fermentative industry for the manufacturing of sake, soy sauce and miso, have identi¢ed genes such as palBory (homologous to A. nidulans palB) that is involved in the alkaline pHmediated signal transduction pathway by activation of PacC transcription factor, and in yeast, CPL1, a calpainlike protease (homolog of palBory ) required for alkaline pH adaptation and sporulation by regulation of RIM101 (a PacC homolog) [8,9]. However, as yet, protein phosphatase genes have not been characterized from A. oryzae. We report for the ¢rst time a gene encoding calmodulin-dependent protein phosphatase from A. oryzae and demonstrate its complementation in a S. cerevisiae calcineurin A disruptant strain.
0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 3 9 8 - 6
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sequencer using the Thermo Sequenase £uorescent labelled primer cycle sequencing kit.
2.1. Strains, plasmids and culture conditions The genomic DNA and cDNA libraries was derived from A. oryzae (RIB40). For cloning and subcloning experiments Escherichia coli P2392, the phage vector VDASH II and pBluescript II SK+ vector were utilized. S. cerevisiae DHT 22-1a, in which both the genes encoding calcineurin A were disrupted (ade2 trp1 leu2 his3 ura3 cmp1 : :LEU2 cmp2: :HIS3) (donated by Prof. T. Miyakawa, Hiroshima University), was used as a host for the expression of A. oryzae cnaA cDNA. The yeast expression vector pYES2 (Invitrogen, The Netherlands) that contained a GAL1 promoter and a URA3 marker was used for subcloning of A. oryzae cnaA cDNA. Czapex-Dox medium supplemented with 2% glucose was used for culturing A. oryzae RIB40. S. cerevisiae was cultured in YPD medium (1% yeast extract, 2% polypeptone and 2% glucose) and YNBD medium (0.67% yeast nitrogen base without amino acids (Difco, USA) and 2% glucose). For yeast complementation experiment YNBD medium plates supplemented with adenine (20 Wg ml31 ) and tryptophan (20 Wg ml31 ) (for selection of transformants) and YPGal (1% yeast extract, 2% polypeptone and 2% galactose; pH 6.5) medium alone or supplemented with 1.2 M NaCl or at pH 8.5 and agar (2%) were used. The transformation of E. coli and S. cerevisiae was performed as described [10,11]. All standard molecular biological procedures were performed according to Sambrook et al. [12]. 2.2. Strategy for the cloning and sequencing of A. oryzae cnaA In order to clone the calcineurin A encoding gene from A. oryzae, two oligonucleotide primers Cn-f (5P-GCG ACT ATG TTG ACA GAG GTT A-3P) and Cn-r (5PGAC GGA TAT TCA TCA CGT TGT T-3P) were designed based on the homologous nucleotide sequence in the A. oryzae EST database. Using A. oryzae genomic DNA as template and Cn-f and Cn-r primers, a 700-bp fragment was ampli¢ed by PCR. This 700-bp fragment was cloned using pT7Blue vector and the resultant plasmid DNA from the recombinant clones was sequenced to con¢rm the sequence similarity to calcineurin A genes from other organisms. Plaque hybridization was performed using the 700-bp fragment as probe and among approximately 5U104 plaques of the VDASH II genomic library screened, 18 positive clones were identi¢ed. The phage DNA was isolated and subjected to restriction enzyme digestion, followed by Southern analysis with the probe. The probe hybridized with a single 7-kb SacI fragment, which was subcloned using pBluescript II SK+ vector and its nucleotide sequence was determined on both strands on a Shimadzu DSQ-2000L automated £uorescent
2.3. Cloning of A. oryzae cnaA cDNA A full length cDNA encoding cnaA from A. oryzae was constructed using the primers CaNN (based on the N-terminal nucleotide sequence) (5P-TAG AGC TCA TGG AAG ATG GCA CTC AGG TG-3P) and CaNC (based on the C-terminal nucleotide sequence) (5P-TGT TCT AGA TTA CAT GCT AAT CCG TCG AGC-3P). SacI and XbaI restriction enzyme sites were introduced at the N-terminal and C-terminal ends of the cDNA for the purpose of subcloning into pBluescript II SK+ and pYES2. The 1.5-kb SacI and XbaI fragment ampli¢ed by PCR contained the full length cnaA cDNA. 2.4. Yeast complementation To facilitate the expression of A. oryzae cnaA in S. cerevisiae, the cnaA cDNA ampli¢ed by PCR was inserted into the SacI and XbaI sites on the 2 W yeast expression vector pYES2 under the control of GAL1 promoter to generate the plasmid pYcnaA. The S. cerevisiae calcineurin A disruptant strain DHT 22-1a (vcmp1 vcmp2) was transformed with pYcnaA and pYES2. Media used for yeast complementation and selection of transformants harboring pYcnaA are as described in Section 2.1. The transformants were grown in the presence of high salt concentrations (1.2 M NaCl) and also under alkaline pH conditions (pH 8.5) to verify the complementation of A. oryzae cnaA in the S. cerevisiae calcineurin A disruptant strain. 3. Results and discussion 3.1. Cloning of the calcineurin A gene and its complementary DNA from A. oryzae Cloning of the calcineurin A gene from A. oryzae was accomplished by designing the sense (Cn-f) and antisense (Cn-r) primers based on the calcineurin A homologous nucleotide sequence data available in the A. oryzae EST database. The 700-bp PCR-ampli¢ed fragment was used as a hybridization probe to screen the A. oryzae VDASH II genomic library as described in Section 2.2. Southern analysis resulted in the identi¢cation of a single 7-kb SacIdigested DNA fragment that contained the entire gene encoding calcineurin A from A. oryzae and was designated as cnaA (Fig. 1). Further, a 1.5-kb cDNA fragment encoding cnaA was also obtained by utilizing the A. oryzae cDNA library as template and performing PCR using the primers CaNN and CaNC which was subcloned into both pBluescript II SK+ vector and the yeast expression
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Fig. 1. Restriction endonuclease map of the DNA fragment containing cnaA. The positions of protein coding regions are indicated by solid boxes. The direction of translation is shown by the arrow. Sites of the representative restriction endonucleases are shown.
Fig. 2. Nucleotide sequence of A. oryzae cnaA. The deduced amino acid sequence of the calcineurin A protein is indicated below the respective codons. Numbers on the left and right margins indicate the position relative to the `A' nucleotide of the start codon and amino acid, respectively. The start codon (ATG) and stop codon (TAA) are in bold. Lower case letters indicate the nucleotides of the introns. The putative `CAAT' and polyadenylation signal sequences are boxed. The amino acid residues in the calmodulin binding domain are shown in bold. The nucleotide sequence data reported have been deposited in the DDBJ nucleotide sequence database under accession number AB063317.
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vector, pYES2, for sequence analysis and yeast complementation experiments, respectively. 3.2. Nucleotide and amino acid sequence analysis of cnaA Nucleotide sequence analysis of cnaA genomic DNA and cDNA revealed that the open reading frame (ORF) extended to 1727 bp and included three introns whose positions were conserved with reference to calcineurin A from A. nidulans. However, in contrast to calcineurin A from A. nidulans [6], which contained four introns, it may be noted that cnaA from A. oryzae had three introns and encoded a putative protein product of 514 amino acids (Fig. 2). Each intron had a 5P GT and 3P AG splice junction, characteristic of eukaryotic introns. The cnaA nucleotide sequence is deposited in the DDBJ nucleotide sequence database under the accession number AB063317.
The deduced amino acid sequence revealed that a large portion of the ORF was homologous to calcineurin A from A. nidulans (92%), N. crassa (84%), human (67%), S. cerevisiae (58%) and Schizosaccharomyces pombe (54%) (Fig. 3). A comparative analysis of amino acid sequence of calcineurin A from A. oryzae and other ¢lamentous fungi showed ¢ve important regions to be highly conserved. These included ¢rstly, the N-terminal half from residues 38 to 70 whose function is unknown [1], secondly, a broad stretch of residues from 71 to 325 which included the catalytic domain because of its homology to other kinds of protein phosphatases [13], thirdly, an adjacent region from residues 326 to 367 that could either be contributing to the binding of calcineurin regulatory subunit or for determinants essential for calmodulin binding to the fourth highly conserved region called the calmodulin binding domain (from residues 400 to 424) [14,15]. Finally the
Fig. 3. Comparison of the deduced amino acid sequence of A. oryzae calcineurin A. Dashes indicate gaps introduced to maintain alignment. Perfectly conserved and well-conserved positions in the alignment are indicated as * and :, respectively. Regions I, II, III represent the calcineurin B binding region, calmodulin binding domain and the auto-inhibitory domain, respectively. Alignment was performed using the ClustalW program supported by DDBJ database. Homology of A. oryzae calcineurin A is indicated as percentage values in the brackets.
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remaining C-terminal region residues from 454 to 478 could be a part of the auto-inhibitory domain which is cleaved upon calmodulin binding and subsequent activation of cnaA [16]. It may also be observed that the carboxy-terminal domain shows lower sequence homology overall. In addition, the 5P and 3P £anking regions of A. oryzae cnaA were analyzed for the presence of a TATA-like box and putative polyadenylation signal sequences, however, no TATA canonical sequences were found. This is not an unusual characteristic of fungal genes. Two sequences ^ATAAA positioned at +103 and +203 were found on the 3P non-coding region and two putative ^CAAT sequences were found at positions 358 and 382 on the 5P non-coding region of cnaA. 3.3. A. oryzae cnaA complemented the S. cerevisiae calcineurin A disruptant strain The observed structural conservative nature of calcineurin A in a wide range of organisms and also recent reports on the implication of its common role in stressmediated signal transduction pathways prompted us to analyze if cnaA from A. oryzae could function in a calcineurin A disrupted strain of S. cerevisiae. To verify if cnaA
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could replace the requirement of calcineurin A in salt stress and alkaline pH-mediated growth, the S. cerevisiae strain (DHT 22-1a; vcmp1 vcmp2) was transformed with the multicopy yeast expression vector, pYES2, harboring cnaA cDNA, which was fused to a galactose-inducible GAL1 promoter (Fig. 4A). Six transformants that were selected by their growth on YNBD medium supplemented with adenine and tryptophan were inoculated onto YPGal induction medium (pH 6.5) in the presence or in the absence of 1.2 M NaCl. The transformants were also inoculated onto YPGal medium (pH 8.5) in order to verify if the replacement of cnaA could make them resistant to alkaline pH conditions. A transformant harboring only the vector pYES2 was selected as control. All the transformants including the control transformant grew well by the end of 3 days on YPGal medium (pH 6.5) (Fig. 4B). However, it may be observed that at pH 8.5 (Fig. 4C) and in the presence of 1.2 M NaCl (Fig. 4D), only the transformants containing cnaA could alone grow in comparison to the control transformant. Such tolerant behavior of the transformants containing cnaA in comparison to the control revealed that A. oryzae cnaA was functional and could complement a calcineurin A disruptant strain of S. cerevisiae. The obtained result suggested a functional homology
Fig. 4. A. oryzae cnaA complemented the salt and alkaline sensitivity of S. cerevisiae (vcmp1 vcmp2). The 1.5-kb SacI^XbaI fragment encoding the A. oryzae calcineurin A cDNA was inserted into the SacI/XbaI site of yeast expression plasmid pYES2, yielding pYcnaA (A). Calcineurin A disruptant strain of S. cerevisiae (vcmp1 vcmp2) transformed with pYES2-derived plasmids (pYES2, pYcnaA) was grown at 30³C on bu¡ered YPGal medium plates containing 2% galactose as a carbon source at pH 6.5 and pH 8.5, respectively (B,C). The strains were also grown in the presence 1.2 M NaCl (D) as described in Section 2. The plates were incubated for 3 days. Plating of the transformants is as shown in the adjacent circle.
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between A. oryzae cnaA and the yeast calcineurin A genes and recon¢rmed the role for calcineurin A in salt stress and alkaline pH-mediated growth of S. cerevisiae. Although we have analyzed the molecular structure of the calcineurin A encoding gene from A. oryzae, we are yet to study its function in detail. While earlier reports of calcineurin A from ¢lamentous fungi suggested its requirement for hyphal growth and cell cycle regulation [5,6], a putative role of this protein phosphatase in sporulation, salt stress response and the alkaline pH-mediated signal transduction pathway in fungi has not been examined. Interestingly, a relation between the control of dimorphism and the pH response pathway has been proposed for the fungal pathogen Candida albicans [17] and since earlier literature has implicated Ca2 /calmodulin-mediated signal transduction processes in fungal morphogenesis [18], it seems reasonable to investigate the possible role of calcineurin in regulation of alkaline pH-mediated growth of ¢lamentous fungi. Moreover, A. oryzae by virtue of its ability to produce very useful enzymes and proteins has been exploited by the Japanese fermentative industry and it would be even more signi¢cant if strains that can adapt to high pH conditions are obtained. The present study assumes signi¢cance since this is the ¢rst report on a gene encoding a protein phosphatase from A. oryzae. It would thus be interesting to study if there is any cross-talk between cnaA and the pal set of genes which are expressed under alkaline pH conditions in this fungi, as it is known to regulate the activation/inactivation of several intracellular substrates by dephosphorylation. We are presently studying cnaA expression under the control of amyB and alcA promoters to enable the understanding of its physiological role in A. oryzae. Acknowledgements The authors gratefully acknowledge the ¢nancial support received from the Japan Society for Promotion of Science through a Grant-in-Aid to Prof. Katsuhiko Kitamoto and a Postdoctoral fellowship to P.R.J. References [1] Kincaid, R. (1993) Calmodulin-dependent protein phosphatases from microorganisms to man. A study in structural conservatism and biological diversity. Adv. Second. Messenger Phosphoprotein Res. 27, 1^ 23.
[2] Sugiura, R., Sio, S.O., Shuntoh, H. and Kuno, T. (2001) Molecular genetic analysis of the calcineurin signaling pathways. Cell. Mol. Life Sci. 58, 278^288. [3] Rusnak, F. and Mertz, P. (2000) Calcineurin: form and function. Physiol. Rev. 4, 1483^1521. [4] Cruz, M.C., Fox, D.S. and Heitman, J. (2001) Calcineurin is required for hyphal elongation during mating and haploid fruiting in Cryptococcus neoformans. EMBO J. 20, 1020^1032. [5] Prokisch, H., Yarden, O., Dieminger, M., Tropschug, M. and Barthelmess, I.B. (1997) Impairment of calcineurin function in Neurospora crassa reveals its essential role in hyphal growth, morphology and maintenance of the apical Ca2 gradient. Mol. Gen. Genet. 256, 104^114. [6] Rasmussen, C., Garen, C., Brining, S., Kincaid, R.L., Means, R.L. and Means, A.R. (1994) The calmodulin-dependent protein phosphatase catalytic subunit (calcineurin A) is an essential gene in Aspergillus nidulans. EMBO J. 13, 3917^3924. [7] Jayashree, T., Praveen Rao, J. and Subramanyam, C. (2000) Regulation of a£atoxin production by Ca2 /calmodulin-dependent protein phosphorylation and dephosphorylation. FEMS Microbiol. Lett. 183, 215^219. [8] Futai, E., Sorimachi, H., Jeong, S.-Y., Kitamoto, K., Ishiura, S. and Suzuki, K. (1999) Aspergillus oryzae pal Bory encodes a calpain-like protease: Homology to Emericella nidulans PalB and conservation of functional regions. J. Biosci. Bioeng. 88, 438^440. [9] Futai, E., Maeda, T., Sorimachi, H., Kitamoto, K., Ishiura, S. and Suzuki, K. (1999) The protease activity of a calpain-like cysteine protease in Saccharomyces cerevisiae is required for alkaline adaptation and sporulation. Mol. Gen. Genet. 260, 559^568. [10] Hanahan, D. (1983) Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166, 577^580. [11] Ito, H., Fukuda, Y., Murata, K. and Kimura, A. (1983) Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153, 163^168. [12] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY [13] Kincaid, R.L., Nightingale, M.S. and Martin, B.M. (1988) Characterization of a cDNA clone encoding the calmodulin-binding domain of mouse brain calcineurin. Proc. Natl. Acad. Sci. USA 85, 8983^ 8987. [14] Higuchi, S., Tamura, J., Giri, P.R., Polli, J.W. and Kincaid, R.L. (1991) Calmodulin-dependent protein phosphatase from Neurospora crassa. Molecular cloning and expression of recombinant catalytic subunit. J. Biol. Chem. 266, 18104^18112. [15] Guerini, D. and Klee, C.B. (1989) Cloning of human calcineurin A: evidence for two isozymes and identi¢cation of a polyproline structural domain. Proc. Natl. Acad. Sci. USA 86, 9183^9187. [16] Hashimoto, Y., Perrino, B.A. and Soderling, T.R. (1990) Identi¢cation of an autoinhibitory domain in calcineurin. J. Biol. Chem. 265, 1924^1927. [17] Ramon, A.M., Porta, A. and Fonzi, W.A. (1999) E¡ect of environmental pH on the morphological development of Candida albicans is mediated via the PacC-related transcription factor encoded by PRR2. J. Bacteriol. 181, 7524^7530. [18] Praveen Rao, J., Reena, G. and Subramanyam, C. (1997) Calmodulin-dependent protein phosphorylation during conidial germination and growth of Neurospora crassa. Mycol. Res. 101, 1484^1488.
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Cloning and sequence analysis of cnaA gene encoding the catalytic subunit of calcineurin from Aspergillus oryzae Praveen Rao Juvvadi, Manabu Arioka, Harushi Nakajima, Katsuhiko Kitamoto * Department of Biotechnology, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-Ku, 113-8657 Tokyo, Japan Received 26 June 2001; received in revised form 12 August 2001; accepted 14 August 2001 First published online 26 September 2001
Abstract Calcineurin has been implicated in ion-homeostasis, stress adaptation in yeast and for hyphal growth in filamentous fungi. Genomic DNA and cDNA encoding the catalytic subunit of calcineurin (cnaA) were isolated from Aspergillus oryzae. The cnaA open reading frame extended to 1727 bp and encoded a putative protein of 514 amino acids. Comparative analysis of the nucleotide sequence of cnaA genomic DNA and cDNA confirmed the presence of three introns and a highly conserved calmodulin binding domain. The deduced amino acid sequence was homologous to calcineurin A from Aspergillus nidulans (92%), Neurospora crassa (84%), human (67%), Saccharomyces cerevisiae (58%) and Schizosaccharomyces pombe (54%). Further, A. oryzae cnaA cDNA complemented S. cerevisiae calcineurin disruptant strain (vcmp1 vcmp2), which was not viable in the presence of high concentrations of NaCl (1.2 M) and at alkaline pH 8.5. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Calcineurin A; Protein phosphatase 2B; Calmodulin-dependent protein phosphatase; Aspergillus oryzae
1. Introduction Calcineurin, the only Ca2 /calmodulin-dependent protein phosphatase (serine/threonine protein phosphatase 2B) recognized as yet in fungi, is a heterodimer comprised of two subunits, viz. K-catalytic subunit A (58^64 kDa) that binds to calmodulin and a L-regulatory subunit B (19 kDa) which is structurally similar to calmodulin. The highly conserved nature and ubiquitous presence of this protein phosphatase throughout the animal and plant kingdom have prompted several investigations into its structure and function [1]. While in mammalian tissues its importance has been implicated in neuronal metabolism, T-lymphocyte proliferation and immune suppression [2], current data in Saccharomyces cerevisiae suggest its
* Corresponding author. Tel. : +81 (3) 5841-5161; Fax: +81 (3) 5841-8033. E-mail address : [email protected] (K. Kitamoto).
requirement for ion-homeostasis, salt stress adaptation, recovery from pheromone-induced growth arrest and in the regulation of alkaline pH-mediated growth [3]. In the basidiomycetous pathogenic fungi Cryptococcus neoformans calcineurin regulates mating, virulence and is required for growth at high temperatures [4]. Among the ¢lamentous fungi its relevance has been reported for hyphal extension in Neurospora crassa, cell cycle regulation in Aspergillus nidulans and secondary metabolism in Aspergillus parasiticus [5^7]. Molecular studies in Aspergillus oryzae, the koji mold, an important fungus widely used in Japanese fermentative industry for the manufacturing of sake, soy sauce and miso, have identi¢ed genes such as palBory (homologous to A. nidulans palB) that is involved in the alkaline pHmediated signal transduction pathway by activation of PacC transcription factor, and in yeast, CPL1, a calpainlike protease (homolog of palBory ) required for alkaline pH adaptation and sporulation by regulation of RIM101 (a PacC homolog) [8,9]. However, as yet, protein phosphatase genes have not been characterized from A. oryzae. We report for the ¢rst time a gene encoding calmodulin-dependent protein phosphatase from A. oryzae and demonstrate its complementation in a S. cerevisiae calcineurin A disruptant strain.
0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 3 9 8 - 6
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sequencer using the Thermo Sequenase £uorescent labelled primer cycle sequencing kit.
2.1. Strains, plasmids and culture conditions The genomic DNA and cDNA libraries was derived from A. oryzae (RIB40). For cloning and subcloning experiments Escherichia coli P2392, the phage vector VDASH II and pBluescript II SK+ vector were utilized. S. cerevisiae DHT 22-1a, in which both the genes encoding calcineurin A were disrupted (ade2 trp1 leu2 his3 ura3 cmp1 : :LEU2 cmp2: :HIS3) (donated by Prof. T. Miyakawa, Hiroshima University), was used as a host for the expression of A. oryzae cnaA cDNA. The yeast expression vector pYES2 (Invitrogen, The Netherlands) that contained a GAL1 promoter and a URA3 marker was used for subcloning of A. oryzae cnaA cDNA. Czapex-Dox medium supplemented with 2% glucose was used for culturing A. oryzae RIB40. S. cerevisiae was cultured in YPD medium (1% yeast extract, 2% polypeptone and 2% glucose) and YNBD medium (0.67% yeast nitrogen base without amino acids (Difco, USA) and 2% glucose). For yeast complementation experiment YNBD medium plates supplemented with adenine (20 Wg ml31 ) and tryptophan (20 Wg ml31 ) (for selection of transformants) and YPGal (1% yeast extract, 2% polypeptone and 2% galactose; pH 6.5) medium alone or supplemented with 1.2 M NaCl or at pH 8.5 and agar (2%) were used. The transformation of E. coli and S. cerevisiae was performed as described [10,11]. All standard molecular biological procedures were performed according to Sambrook et al. [12]. 2.2. Strategy for the cloning and sequencing of A. oryzae cnaA In order to clone the calcineurin A encoding gene from A. oryzae, two oligonucleotide primers Cn-f (5P-GCG ACT ATG TTG ACA GAG GTT A-3P) and Cn-r (5PGAC GGA TAT TCA TCA CGT TGT T-3P) were designed based on the homologous nucleotide sequence in the A. oryzae EST database. Using A. oryzae genomic DNA as template and Cn-f and Cn-r primers, a 700-bp fragment was ampli¢ed by PCR. This 700-bp fragment was cloned using pT7Blue vector and the resultant plasmid DNA from the recombinant clones was sequenced to con¢rm the sequence similarity to calcineurin A genes from other organisms. Plaque hybridization was performed using the 700-bp fragment as probe and among approximately 5U104 plaques of the VDASH II genomic library screened, 18 positive clones were identi¢ed. The phage DNA was isolated and subjected to restriction enzyme digestion, followed by Southern analysis with the probe. The probe hybridized with a single 7-kb SacI fragment, which was subcloned using pBluescript II SK+ vector and its nucleotide sequence was determined on both strands on a Shimadzu DSQ-2000L automated £uorescent
2.3. Cloning of A. oryzae cnaA cDNA A full length cDNA encoding cnaA from A. oryzae was constructed using the primers CaNN (based on the N-terminal nucleotide sequence) (5P-TAG AGC TCA TGG AAG ATG GCA CTC AGG TG-3P) and CaNC (based on the C-terminal nucleotide sequence) (5P-TGT TCT AGA TTA CAT GCT AAT CCG TCG AGC-3P). SacI and XbaI restriction enzyme sites were introduced at the N-terminal and C-terminal ends of the cDNA for the purpose of subcloning into pBluescript II SK+ and pYES2. The 1.5-kb SacI and XbaI fragment ampli¢ed by PCR contained the full length cnaA cDNA. 2.4. Yeast complementation To facilitate the expression of A. oryzae cnaA in S. cerevisiae, the cnaA cDNA ampli¢ed by PCR was inserted into the SacI and XbaI sites on the 2 W yeast expression vector pYES2 under the control of GAL1 promoter to generate the plasmid pYcnaA. The S. cerevisiae calcineurin A disruptant strain DHT 22-1a (vcmp1 vcmp2) was transformed with pYcnaA and pYES2. Media used for yeast complementation and selection of transformants harboring pYcnaA are as described in Section 2.1. The transformants were grown in the presence of high salt concentrations (1.2 M NaCl) and also under alkaline pH conditions (pH 8.5) to verify the complementation of A. oryzae cnaA in the S. cerevisiae calcineurin A disruptant strain. 3. Results and discussion 3.1. Cloning of the calcineurin A gene and its complementary DNA from A. oryzae Cloning of the calcineurin A gene from A. oryzae was accomplished by designing the sense (Cn-f) and antisense (Cn-r) primers based on the calcineurin A homologous nucleotide sequence data available in the A. oryzae EST database. The 700-bp PCR-ampli¢ed fragment was used as a hybridization probe to screen the A. oryzae VDASH II genomic library as described in Section 2.2. Southern analysis resulted in the identi¢cation of a single 7-kb SacIdigested DNA fragment that contained the entire gene encoding calcineurin A from A. oryzae and was designated as cnaA (Fig. 1). Further, a 1.5-kb cDNA fragment encoding cnaA was also obtained by utilizing the A. oryzae cDNA library as template and performing PCR using the primers CaNN and CaNC which was subcloned into both pBluescript II SK+ vector and the yeast expression
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Fig. 1. Restriction endonuclease map of the DNA fragment containing cnaA. The positions of protein coding regions are indicated by solid boxes. The direction of translation is shown by the arrow. Sites of the representative restriction endonucleases are shown.
Fig. 2. Nucleotide sequence of A. oryzae cnaA. The deduced amino acid sequence of the calcineurin A protein is indicated below the respective codons. Numbers on the left and right margins indicate the position relative to the `A' nucleotide of the start codon and amino acid, respectively. The start codon (ATG) and stop codon (TAA) are in bold. Lower case letters indicate the nucleotides of the introns. The putative `CAAT' and polyadenylation signal sequences are boxed. The amino acid residues in the calmodulin binding domain are shown in bold. The nucleotide sequence data reported have been deposited in the DDBJ nucleotide sequence database under accession number AB063317.
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vector, pYES2, for sequence analysis and yeast complementation experiments, respectively. 3.2. Nucleotide and amino acid sequence analysis of cnaA Nucleotide sequence analysis of cnaA genomic DNA and cDNA revealed that the open reading frame (ORF) extended to 1727 bp and included three introns whose positions were conserved with reference to calcineurin A from A. nidulans. However, in contrast to calcineurin A from A. nidulans [6], which contained four introns, it may be noted that cnaA from A. oryzae had three introns and encoded a putative protein product of 514 amino acids (Fig. 2). Each intron had a 5P GT and 3P AG splice junction, characteristic of eukaryotic introns. The cnaA nucleotide sequence is deposited in the DDBJ nucleotide sequence database under the accession number AB063317.
The deduced amino acid sequence revealed that a large portion of the ORF was homologous to calcineurin A from A. nidulans (92%), N. crassa (84%), human (67%), S. cerevisiae (58%) and Schizosaccharomyces pombe (54%) (Fig. 3). A comparative analysis of amino acid sequence of calcineurin A from A. oryzae and other ¢lamentous fungi showed ¢ve important regions to be highly conserved. These included ¢rstly, the N-terminal half from residues 38 to 70 whose function is unknown [1], secondly, a broad stretch of residues from 71 to 325 which included the catalytic domain because of its homology to other kinds of protein phosphatases [13], thirdly, an adjacent region from residues 326 to 367 that could either be contributing to the binding of calcineurin regulatory subunit or for determinants essential for calmodulin binding to the fourth highly conserved region called the calmodulin binding domain (from residues 400 to 424) [14,15]. Finally the
Fig. 3. Comparison of the deduced amino acid sequence of A. oryzae calcineurin A. Dashes indicate gaps introduced to maintain alignment. Perfectly conserved and well-conserved positions in the alignment are indicated as * and :, respectively. Regions I, II, III represent the calcineurin B binding region, calmodulin binding domain and the auto-inhibitory domain, respectively. Alignment was performed using the ClustalW program supported by DDBJ database. Homology of A. oryzae calcineurin A is indicated as percentage values in the brackets.
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remaining C-terminal region residues from 454 to 478 could be a part of the auto-inhibitory domain which is cleaved upon calmodulin binding and subsequent activation of cnaA [16]. It may also be observed that the carboxy-terminal domain shows lower sequence homology overall. In addition, the 5P and 3P £anking regions of A. oryzae cnaA were analyzed for the presence of a TATA-like box and putative polyadenylation signal sequences, however, no TATA canonical sequences were found. This is not an unusual characteristic of fungal genes. Two sequences ^ATAAA positioned at +103 and +203 were found on the 3P non-coding region and two putative ^CAAT sequences were found at positions 358 and 382 on the 5P non-coding region of cnaA. 3.3. A. oryzae cnaA complemented the S. cerevisiae calcineurin A disruptant strain The observed structural conservative nature of calcineurin A in a wide range of organisms and also recent reports on the implication of its common role in stressmediated signal transduction pathways prompted us to analyze if cnaA from A. oryzae could function in a calcineurin A disrupted strain of S. cerevisiae. To verify if cnaA
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could replace the requirement of calcineurin A in salt stress and alkaline pH-mediated growth, the S. cerevisiae strain (DHT 22-1a; vcmp1 vcmp2) was transformed with the multicopy yeast expression vector, pYES2, harboring cnaA cDNA, which was fused to a galactose-inducible GAL1 promoter (Fig. 4A). Six transformants that were selected by their growth on YNBD medium supplemented with adenine and tryptophan were inoculated onto YPGal induction medium (pH 6.5) in the presence or in the absence of 1.2 M NaCl. The transformants were also inoculated onto YPGal medium (pH 8.5) in order to verify if the replacement of cnaA could make them resistant to alkaline pH conditions. A transformant harboring only the vector pYES2 was selected as control. All the transformants including the control transformant grew well by the end of 3 days on YPGal medium (pH 6.5) (Fig. 4B). However, it may be observed that at pH 8.5 (Fig. 4C) and in the presence of 1.2 M NaCl (Fig. 4D), only the transformants containing cnaA could alone grow in comparison to the control transformant. Such tolerant behavior of the transformants containing cnaA in comparison to the control revealed that A. oryzae cnaA was functional and could complement a calcineurin A disruptant strain of S. cerevisiae. The obtained result suggested a functional homology
Fig. 4. A. oryzae cnaA complemented the salt and alkaline sensitivity of S. cerevisiae (vcmp1 vcmp2). The 1.5-kb SacI^XbaI fragment encoding the A. oryzae calcineurin A cDNA was inserted into the SacI/XbaI site of yeast expression plasmid pYES2, yielding pYcnaA (A). Calcineurin A disruptant strain of S. cerevisiae (vcmp1 vcmp2) transformed with pYES2-derived plasmids (pYES2, pYcnaA) was grown at 30³C on bu¡ered YPGal medium plates containing 2% galactose as a carbon source at pH 6.5 and pH 8.5, respectively (B,C). The strains were also grown in the presence 1.2 M NaCl (D) as described in Section 2. The plates were incubated for 3 days. Plating of the transformants is as shown in the adjacent circle.
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between A. oryzae cnaA and the yeast calcineurin A genes and recon¢rmed the role for calcineurin A in salt stress and alkaline pH-mediated growth of S. cerevisiae. Although we have analyzed the molecular structure of the calcineurin A encoding gene from A. oryzae, we are yet to study its function in detail. While earlier reports of calcineurin A from ¢lamentous fungi suggested its requirement for hyphal growth and cell cycle regulation [5,6], a putative role of this protein phosphatase in sporulation, salt stress response and the alkaline pH-mediated signal transduction pathway in fungi has not been examined. Interestingly, a relation between the control of dimorphism and the pH response pathway has been proposed for the fungal pathogen Candida albicans [17] and since earlier literature has implicated Ca2 /calmodulin-mediated signal transduction processes in fungal morphogenesis [18], it seems reasonable to investigate the possible role of calcineurin in regulation of alkaline pH-mediated growth of ¢lamentous fungi. Moreover, A. oryzae by virtue of its ability to produce very useful enzymes and proteins has been exploited by the Japanese fermentative industry and it would be even more signi¢cant if strains that can adapt to high pH conditions are obtained. The present study assumes signi¢cance since this is the ¢rst report on a gene encoding a protein phosphatase from A. oryzae. It would thus be interesting to study if there is any cross-talk between cnaA and the pal set of genes which are expressed under alkaline pH conditions in this fungi, as it is known to regulate the activation/inactivation of several intracellular substrates by dephosphorylation. We are presently studying cnaA expression under the control of amyB and alcA promoters to enable the understanding of its physiological role in A. oryzae. Acknowledgements The authors gratefully acknowledge the ¢nancial support received from the Japan Society for Promotion of Science through a Grant-in-Aid to Prof. Katsuhiko Kitamoto and a Postdoctoral fellowship to P.R.J. References [1] Kincaid, R. (1993) Calmodulin-dependent protein phosphatases from microorganisms to man. A study in structural conservatism and biological diversity. Adv. Second. Messenger Phosphoprotein Res. 27, 1^ 23.
[2] Sugiura, R., Sio, S.O., Shuntoh, H. and Kuno, T. (2001) Molecular genetic analysis of the calcineurin signaling pathways. Cell. Mol. Life Sci. 58, 278^288. [3] Rusnak, F. and Mertz, P. (2000) Calcineurin: form and function. Physiol. Rev. 4, 1483^1521. [4] Cruz, M.C., Fox, D.S. and Heitman, J. (2001) Calcineurin is required for hyphal elongation during mating and haploid fruiting in Cryptococcus neoformans. EMBO J. 20, 1020^1032. [5] Prokisch, H., Yarden, O., Dieminger, M., Tropschug, M. and Barthelmess, I.B. (1997) Impairment of calcineurin function in Neurospora crassa reveals its essential role in hyphal growth, morphology and maintenance of the apical Ca2 gradient. Mol. Gen. Genet. 256, 104^114. [6] Rasmussen, C., Garen, C., Brining, S., Kincaid, R.L., Means, R.L. and Means, A.R. (1994) The calmodulin-dependent protein phosphatase catalytic subunit (calcineurin A) is an essential gene in Aspergillus nidulans. EMBO J. 13, 3917^3924. [7] Jayashree, T., Praveen Rao, J. and Subramanyam, C. (2000) Regulation of a£atoxin production by Ca2 /calmodulin-dependent protein phosphorylation and dephosphorylation. FEMS Microbiol. Lett. 183, 215^219. [8] Futai, E., Sorimachi, H., Jeong, S.-Y., Kitamoto, K., Ishiura, S. and Suzuki, K. (1999) Aspergillus oryzae pal Bory encodes a calpain-like protease: Homology to Emericella nidulans PalB and conservation of functional regions. J. Biosci. Bioeng. 88, 438^440. [9] Futai, E., Maeda, T., Sorimachi, H., Kitamoto, K., Ishiura, S. and Suzuki, K. (1999) The protease activity of a calpain-like cysteine protease in Saccharomyces cerevisiae is required for alkaline adaptation and sporulation. Mol. Gen. Genet. 260, 559^568. [10] Hanahan, D. (1983) Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166, 577^580. [11] Ito, H., Fukuda, Y., Murata, K. and Kimura, A. (1983) Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153, 163^168. [12] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY [13] Kincaid, R.L., Nightingale, M.S. and Martin, B.M. (1988) Characterization of a cDNA clone encoding the calmodulin-binding domain of mouse brain calcineurin. Proc. Natl. Acad. Sci. USA 85, 8983^ 8987. [14] Higuchi, S., Tamura, J., Giri, P.R., Polli, J.W. and Kincaid, R.L. (1991) Calmodulin-dependent protein phosphatase from Neurospora crassa. Molecular cloning and expression of recombinant catalytic subunit. J. Biol. Chem. 266, 18104^18112. [15] Guerini, D. and Klee, C.B. (1989) Cloning of human calcineurin A: evidence for two isozymes and identi¢cation of a polyproline structural domain. Proc. Natl. Acad. Sci. USA 86, 9183^9187. [16] Hashimoto, Y., Perrino, B.A. and Soderling, T.R. (1990) Identi¢cation of an autoinhibitory domain in calcineurin. J. Biol. Chem. 265, 1924^1927. [17] Ramon, A.M., Porta, A. and Fonzi, W.A. (1999) E¡ect of environmental pH on the morphological development of Candida albicans is mediated via the PacC-related transcription factor encoded by PRR2. J. Bacteriol. 181, 7524^7530. [18] Praveen Rao, J., Reena, G. and Subramanyam, C. (1997) Calmodulin-dependent protein phosphorylation during conidial germination and growth of Neurospora crassa. Mycol. Res. 101, 1484^1488.
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