- Email: [email protected]
Cloning and characterization of the phenylalanyl-tRNA synthetase β subunit gene from Candida albicans
FEMS Microbiology Letters 161 (1998) 179^185
Cloning and characterization of the phenylalanyl-tRNA synthetase L subunit gene from Candida albicans Antonio Marcilla *, Claudia Pallotti, Maria Gomez-Lobo, Pedro Caballero, Eulogio Valentin, Rafael Sentandreu Seccioè Departamental de Microbiologia, Facultat de Farmacia, Universitat de Valencia, Avgda. Vicent Andreès Estelleès, s/n, 46100 Burjassot (Valencia), Spain Received 21 January 1998; revised 9 February 1998; accepted 9 February 1998
Abstract A Candida albicans expression library was constructed from RNA isolated from regenerating protoplasts. A 1.4-kb cDNA clone was used to isolate a genomic fragment. Sequence analysis revealed an open reading frame of 593 amino acids with an overall identity of 63.6% with the phenylalanyl-tRNA synthetase L subunit (FRS1) of Saccharomyces cerevisiae. We named it CaFRS1. It is located in a single copy in chromosome R, SfiI fragment M. Its expression showed a decrease during the cell wall regeneration process in protoplasts of both yeast and mycelial cells of C. albicans, suggesting its requirement thereof in initial steps of the cell wall synthesis. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. Keywords : Phenylalanyl-tRNA synthetase ; Regenerating protoplast; Candida albicans
1. Introduction Candida albicans is an opportunistic pathogenic fungus that produces infections in humans with increasing incidence, mainly in immunocompromised patients [1]. This fungus can grow as yeast and/or mycelial cells depending on environmental conditions, this morphological transition being associated with its pathogenicity [1^3]. Amino acyl-tRNA synthetases are essential enzymes which activate amino acids for incorporation into growing polypeptide chains in protein synthesis. Recent studies have proposed their participation in * Corresponding author. Tel.: +34 (6) 3864299; Fax: +34 (6) 3864682; E-mail: [email protected]
phosphorylation cascades originated by the cellular adaptation to environmental changes [4]. Although synthetases from eukaryotic organisms are larger than those from prokaryotic enzymes, they share certain sets of sequence motifs [5]. Unlike most amino acyl-tRNA synthetases, phenylalanyltRNA synthetases exhibit a tetrameric structure (K2 L2 ) in prokaryotic organisms as well as in the cytosolic enzymes of yeast and mammalian cells [6]. In an approach to clone genes encoding proteins involved in the construction of the cell wall of C. albicans, we searched for genes encoding proteins synthesized de novo during protoplast regeneration [7]. In this study evidence of a phenylalanyl-tRNA synthetase of C. albicans synthesized de novo is presented.
0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 0 7 2 - X
FEMSLE 8082 19-3-98
180
A. Marcilla et al. / FEMS Microbiology Letters 161 (1998) 179^185
FEMSLE 8082 19-3-98
A. Marcilla et al. / FEMS Microbiology Letters 161 (1998) 179^185
181
Fig. 1. DNA sequence of CaFRS1 showing the predicted amino acid sequence of the encoded protein. Putative N-glycosylation (8), binding site for tRNAPhe (bold), and ATP-binding sites (boxed) are indicated. The position of the ¢rst nucleotides of the P1 cDNA is underlined.
6
2. Materials and methods
cDNA with digoxigenin and hybridization were performed following the Boehringer Mannheim kit instructions.
2.1. Strains and growth conditions C. albicans ATCC 26555 (serotype A) was employed in this study. It was maintained by subculturing every 2^3 weeks on Sabouraud-dextrose agar and propagated in Lee medium [8]. Escherichia coli DH5K was used routinely for transformations. E. coli NM522 was used as host strain for the phage VExCell (Pharmacia). Standard DNA manipulation techniques were carried out as described [9]. 2.2. cDNA library construction and screening A C. albicans cDNA library in phage expression vector VExCell (Pharmacia) was constructed with RNA isolated from protoplasts regenerating for 3 h as yeast cells. The library contained 1.16U104 clones with an average insert size of 1.5 kb. Protoplasts were obtained and regenerated for 1 h as previously described [10]. For mRNA isolation and cDNA synthesis, mRNA Puri¢cation Kit and cDNA Synthesis Kit, both from Pharmacia, were used according to the manufacturer's instructions. cDNAs obtained were subsequently linked to EcoRI-NotI adapters prior to their insertion into EcoRI-digested VExCell. A commercially available rabbit polyclonal antiserum raised against human phosphatidylinositol 3P kinase (PI3Pk) (Upstate Biotechnology Inc.) [11] was used to screen the cDNA library as previously described [12]. Subsequent fragment isolation and subcloning were done according to established protocols.
2.4. Northern and Southern analysis Protoplasts of C. albicans growing at 28³C or 37³C for varying periods were washed and resuspended in 0.1 M LiCl, 0.01 M EDTA, 0.01 M Tris-HCl, pH 7.4, 0.2% (w/v) sodium dodecyl sulfate (SDS) (LETS bu¡er) containing 0.6 M KCl as osmotic stabilizer. Total RNA isolation and Northern blot were carried out essentially as described [12] using disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2P-(5P-chloro)tricyclo[3,3,1,13;7 ]decan}-4-yl) phenyl phosphate (CSPD, Boehringer Mannheim) as a substrate. Genomic DNA isolation and Southern protocols have been described [14]. 2.5. DNA sequencing and analysis DNA sequencing was performed on both strands by the dideoxy chain-termination method [15] with the AutoRead Sequencing Kit (Pharmacia), and cycle sequencing with the fmol Cycle Sequencing System (Promega), following the suppliers' instructions. Sequencing results were analyzed with the ALF DNA Sequencer (Pharmacia Biotech). Nucleotide sequence analysis was performed with the PROSITE Program [16]. Homology searches of NCBI databases were conducted with the BLAST Program [17,18]. The sequence of the CaFRS1 DNA has been deposited in the EMBL database with accession number Y12589.
2.3. Screening of a genomic library
3. Results and discussion
The isolated cDNA fragment was used as a probe to screen a genomic library constructed from C. albicans C9 chromosomal DNA in the plasmid p1041 [13], as per standard procedures [9]. Labelling of
3.1. Cloning of a cDNA encoding phenylalanyl-tRNA synthetase To identify proteins involved in events related to
FEMSLE 8082 19-3-98
182
A. Marcilla et al. / FEMS Microbiology Letters 161 (1998) 179^185
Fig. 2. Alignment of known phenylalanyl-tRNA synthetase L subunit amino acid sequences. The sequences are from C. albicans (CaFRS1), S. cerevisiae cytosolic enzyme (FRS1) [6], C. elegans (Ce Syf) (GenBank accession number Z50044), and E. coli (Ec Syf) (EMBL accession number K02844). Identical residues are boxed.
FEMSLE 8082 19-3-98
A. Marcilla et al. / FEMS Microbiology Letters 161 (1998) 179^185
Fig. 3. Northern blot analysis of the CaFRS1 transcript. Total RNA (20 Wg) from protoplasts grown for 1, 3, 5 and 7 h at 28³C or 37³C were electrophoresed through an agarose gel and transferred to nylon membranes as described in Section 2. The blotted membrane was hybridized with P1 cDNA probe labelled with digoxigenin (A). The corresponding ethidium bromidestained gel is shown in B. The positions of the 28S and 18S ribosomal RNAs are indicated on the left.
the construction of C. albicans cell wall, we immunoscreened a VExCell cDNA library obtained from regenerating protoplasts, which should synthesize de novo an entire cell wall [7,10]. We hypothesized that messengers encoding proteins involved in cell wall regeneration would be highly represented in this library. Fifteen thousand recombinant clones were immunoscreened with a rabbit polyclonal antiserum raised against human PI3Pk [11]. Several positive clones were isolated after three rounds of immunoscreening, and one of them, designated P1, was shown to carry an insert of 1413 bp. Using this cDNA as a probe, a C. albicans genomic library was screened. Nine thousand colonies were screened as described [9], and four positive clones were puri¢ed after three rounds of screening. One of them, designated PA, contained
183
an insert of 7.2 kb. After sequencing an internal fragment of 2028 bp, which contains the entire cDNA clone P1, a single open reading frame of 593 amino acids, extending from base pair 157 up to a stop codon at position 1936, was found (Fig. 1). These data indicated the absence of introns in this gene, and thus we used the cDNA probe for further genomic analysis. A BLAST search in available databases revealed that the nucleotide sequence of PA was highly similar to the Saccharomyces cerevisiae gene FRS1 encoding for the phenylalanyl-tRNA synthetase (PheRS) L (large) subunit [6]. The deduced amino acid sequence of PA showed 63.6% identity with the cytosolic enzyme of S. cerevisiae, and contains the binding site for tRNAPhe , as occurs in S. cerevisiae [19], as well as the potential binding region to the small subunit (K) to form the K2 L2 structure (Fig. 1), required for activity [20]. Fig. 2 shows the alignment of the deduced amino acid sequences of three known PheRS L subunits (SYF) with CaFRS1. The similarities between the S. cerevisiae and C. albicans proteins reached almost 95% in certain regions (amino acids 124^152; 434^ 469 of S. cerevisiae FRS1) [6]. Signi¢cant similarity was observed with the Caenorhabditis elegans deduced amino acid sequence (44.7% identity) (GenBank Sequence Database accession number Z50044). When compared to bacterial enzymes, CaFRS1 exhibited less similarity, dropping to 8.6% identity with the E. coli enzyme (EMBL Sequence Database accession number K02844) (Fig. 2). This similarity was due to several conserved amino acid residues among all enzymes, which would indicate important roles for those residues in the function of the enzymes. An interesting common feature of the deduced amino acid sequence of CaFRS1 with the cytosolic enzyme of S. cerevisiae is the absence of a region in the C-terminus, which is present in bacterial enzymes [21] (positions 698^794 in E. coli) (Fig. 2), in positions 685^780 in the protein of the bacterium Thermus thermophilus [21], and also in S. cerevisiae mitochondrial enzyme [20]. It has been speculated that this extension confers independence from the cytosolic K subunit [21]. The two yeast cytosolic enzymes share a high number of cysteine residues, three being surrounded by similar amino acids and located in
FEMSLE 8082 19-3-98
184
A. Marcilla et al. / FEMS Microbiology Letters 161 (1998) 179^185
identical positions in the predicted amino acid sequence (C28, C266 and C306 of the CaFRS1 sequence) (Fig. 2). Further studies should uncover the function of these regions, but it is tempting to postulate a role in establishing disul¢de bridges needed for the active quaternary structure. 3.2. Southern blot analysis and mapping of the CaFRS1 gene To obtain information about the genomic organization of the CaFRS1 gene, Southern blot analysis was performed using chromosomal DNA digested with EcoRI, and EcoRI plus KpnI and probed with DIG-labelled P1 cDNA. The 1.4-kb probe hybridized only with an 11-kb band when genomic DNA was digested with EcoRI, and two bands of 7 and 4 kb, respectively, when digested with EcoRI plus KpnI, con¢rming the presence of a KpnI site found in the cDNA (data not shown). The probe failed to hybridize with S. cerevisiae genomic DNA digested with EcoRI (data not shown). Although the similarity with FRS1 of S. cerevisiae is high, the differences in the overall DNA sequence and codon usage in the two species could explain this result [22]. These results imply that the CaFRS1 gene is present in a single copy in the genome of C. albicans. Using the same cDNA probe, the chromosomal localization of the CaFRS1 gene was determined. It is located in chromosome R in fragment M of the physical map of C. albicans obtained by fractionation of chromosomes with S¢I [23], being identi¢ed in the fosmids 14E7 and 17D10 (B.B. Magee, personal communication). 3.3. Expression of CaFRS1 reduces during the regeneration of the cell wall in protoplasts To explore transcription of CaFRS1, total RNA was isolated from C. albicans regenerating protoplasts as yeast (28³C) or mycelial (37³C) cells for di¡erent periods, and analyzed by Northern blot hybridization (Fig. 3). A transcript of approximately 1.8 kb was detected, in agreement with the hypothesized size of the gene in C. albicans and FRS1 in S. cerevisiae [6]. The expression of the CaFRS1 transcript decreased during protoplast regeneration at both
28³C and 37³C, being undetectable after 7 h in protoplasts regenerating in the yeast form (Fig. 3). These data could indicate higher stability of the RNA in protoplasts regenerating at 37³C (mycelial cells), although no signi¢cant di¡erences were observed when total RNA from mature cells was analyzed (data not shown). The high level of expression of CaFRS1 in initial steps of protoplast regeneration would suggest a role for phenylalanyl-tRNA synthetase in regulating the synthesis of proteins involved in cell wall synthesis, one of the ¢rst events to occur in protoplasts [10]. Hence, a dual function for tRNA synthetases has been proposed; in addition to their role in tRNA charging, they could regulate protein synthesis, initiating a protein phosphorylation cascade in response to certain stress situations such as temperature changes or deprivation of amino acids [4]. Future studies should analyze the role of tRNA synthetases in processes such as the dimorphic transition originated by stress situations.
Acknowledgments We are grateful to Dr. B.B. Magee (University of Minnesota) for the genomic mapping of the CaFRS1 gene, the generous gift of the C. albicans C9 genomic library, and critical reading of the manuscript. This work was supported by Fondo de Investigaciones Sanitarias de la Seguridad Social (FIS 95/1602), Spain, CICYT (PM96-0019), Spain, and BIOMED2 (BMH4-CT96-0310) Brussels, EU. C.P. was supported by a doctoral fellowship from the Institute of International Cooperation, Spanish Ministry of Foreign A¡airs. M.G.-L. was supported by a doctoral fellowship from CICYT, Spanish Ministry of Culture and Education.
References [1] Odds, F.C. (1988) Candida and Candidosis. A Review and Bibliography. Bailliere Tindal, London. [2] Calderone, R. and Braun, P.C. (1991) Adherence and receptor relationships of Candida albicans. Microbiol Rev. 55, 1^20. [3] Sentandreu, R., Elorza, M.V., Mormeneo, S., Sanjuaèn, R. and Iranzo, M. (1993) Possible roles of mannoproteins in the construction of Candida albicans cell wall. In : Dimorphic Fungi
FEMSLE 8082 19-3-98
A. Marcilla et al. / FEMS Microbiology Letters 161 (1998) 179^185
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11] [12]
[13]
in Biology and Medicine (Van den Bossche, H., Odds, F. and Kerridge, D., Eds.), pp. 169^175. Plenum Press, New York. Clemens, M.J. (1990) Does protein phosphorylation play a role in translational control by eukaryotic aminoacyl-tRNA synthetases? Trends Biochem. Sci. 15, 172^175. Eriani, G., Delarue, M., Poch, O., Ganglo¡, J. and Moras, D. (1990) Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs. Nature 347, 203^206. Sanni, A., Mirande, M., Ebel, J.-P., Boulanger, Y., Waller, J.-P. and Fasiolo, F. (1988) Structure and expression of the genes encoding the K and L subunits of yeast phenylalanyltRNA synthetase. J. Biol. Chem. 263, 15407^15415. Elorza, M.V., Marcilla, A., Sanjuaèn, R., Mormeneo, S. and Sentandreu, R. (1994) Incorporation of speci¢c wall proteins during yeast and mycelial protoplast regeneration in Candida albicans. Arch. Microbiol. 161, 145^151. Lee, K.L., Buckley, M.R. and Campbell, C. (1975) An amino acid liquid synthetic medium for development of mycelial and yeast forms of Candida albicans. Sabouraudia 13, 148^153. 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. Elorza, M.V., Rico, H., Gozalbo, D. and Sentandreu, R. (1983) Cell wall composition and protoplast regeneration in Candida albicans. Antonie van Leeuwenhoek Int. J. Gen. Microbiol. 49, 457^469. Kapeller, R. and Cantley, L. C. (1994) Phosphatidylinositol 3-kinase. BioEssays 16, 565^576. Ramoèn, A.M., Gil, R., Burgal, M., Sentandreu, R. and Valent|èn, E. (1996) A novel cell wall protein speci¢c to the mycelial form of Yarrowia lipolytica. Yeast 12, 1535^1548. Goshorn, A.K., Grindle, S.M. and Scherer, S. (1992) Gene isolation by complementation in Candida albicans and applications to physical and genetic mapping. Infect. Immun. 60, 876^884.
185
[14] Rose, M.D., Winston, F. and Hieter, P. (1990) Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [15] Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463^5467. [16] Bairoch, A. (1993) The PROSITE dictionary of sites and patterns in proteins, its current status. Nucleic Acids Res. 21, 3097^3103. [17] Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403^410. [18] Gish, W. and States, D.J. (1993) Identi¢cation of protein coding regions by database similarity search. Nature Genet. 3, 266^272. [19] Sanni, A., Walter, P., Boulanger, Y., Ebel, J.-P. and Fasiolo, F. (1991) Evolution of aminoacyl-tRNA synthetase quaternary structure and activity: Saccharomyces cerevisiae mitochondrial phenylalanyl-tRNA synthetase. Proc. Natl. Acad. Sci. USA 88, 8387^8391. [20] Aphasizhev, R., Senger, B., Rengers, J.-U., Sprinz, M., Walter, P., Nussbaum, G. and Fasiolo, F. (1996) Conservation in evolution for a small monomeric phenylalanyl-tRNA synthetase of the tRNAPhe recognition nucleotides and initial aminoacylation site. Biochemistry 35, 117^123. [21] Keller, B., Kast, P. and Hennecke, H. (1992) Cloning and sequence analysis of the phenylalanyl-tRNA synthetase genes (pheST) from Thermus thermophilus. FEBS Lett. 301, 83^ 88. [22] Brown, A.J.P, Bertram, G., Feldmann, P.J., Peggie, M.W. and Swoboda, R.K. (1991) Codon utilisation in the pathogenic yeast Candida albicans. Nucleic Acids Res. 19, 4298. [23] Chu, W.-S., Magee, B.B. and Magee, P.T. (1993) Construction of an S¢I macrorestriction map of the Candida albicans genome. J. Bacteriol. 175, 6637^6651.
FEMSLE 8082 19-3-98
Cloning and characterization of the phenylalanyl-tRNA synthetase L subunit gene from Candida albicans Antonio Marcilla *, Claudia Pallotti, Maria Gomez-Lobo, Pedro Caballero, Eulogio Valentin, Rafael Sentandreu Seccioè Departamental de Microbiologia, Facultat de Farmacia, Universitat de Valencia, Avgda. Vicent Andreès Estelleès, s/n, 46100 Burjassot (Valencia), Spain Received 21 January 1998; revised 9 February 1998; accepted 9 February 1998
Abstract A Candida albicans expression library was constructed from RNA isolated from regenerating protoplasts. A 1.4-kb cDNA clone was used to isolate a genomic fragment. Sequence analysis revealed an open reading frame of 593 amino acids with an overall identity of 63.6% with the phenylalanyl-tRNA synthetase L subunit (FRS1) of Saccharomyces cerevisiae. We named it CaFRS1. It is located in a single copy in chromosome R, SfiI fragment M. Its expression showed a decrease during the cell wall regeneration process in protoplasts of both yeast and mycelial cells of C. albicans, suggesting its requirement thereof in initial steps of the cell wall synthesis. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. Keywords : Phenylalanyl-tRNA synthetase ; Regenerating protoplast; Candida albicans
1. Introduction Candida albicans is an opportunistic pathogenic fungus that produces infections in humans with increasing incidence, mainly in immunocompromised patients [1]. This fungus can grow as yeast and/or mycelial cells depending on environmental conditions, this morphological transition being associated with its pathogenicity [1^3]. Amino acyl-tRNA synthetases are essential enzymes which activate amino acids for incorporation into growing polypeptide chains in protein synthesis. Recent studies have proposed their participation in * Corresponding author. Tel.: +34 (6) 3864299; Fax: +34 (6) 3864682; E-mail: [email protected]
phosphorylation cascades originated by the cellular adaptation to environmental changes [4]. Although synthetases from eukaryotic organisms are larger than those from prokaryotic enzymes, they share certain sets of sequence motifs [5]. Unlike most amino acyl-tRNA synthetases, phenylalanyltRNA synthetases exhibit a tetrameric structure (K2 L2 ) in prokaryotic organisms as well as in the cytosolic enzymes of yeast and mammalian cells [6]. In an approach to clone genes encoding proteins involved in the construction of the cell wall of C. albicans, we searched for genes encoding proteins synthesized de novo during protoplast regeneration [7]. In this study evidence of a phenylalanyl-tRNA synthetase of C. albicans synthesized de novo is presented.
0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 0 7 2 - X
FEMSLE 8082 19-3-98
180
A. Marcilla et al. / FEMS Microbiology Letters 161 (1998) 179^185
FEMSLE 8082 19-3-98
A. Marcilla et al. / FEMS Microbiology Letters 161 (1998) 179^185
181
Fig. 1. DNA sequence of CaFRS1 showing the predicted amino acid sequence of the encoded protein. Putative N-glycosylation (8), binding site for tRNAPhe (bold), and ATP-binding sites (boxed) are indicated. The position of the ¢rst nucleotides of the P1 cDNA is underlined.
6
2. Materials and methods
cDNA with digoxigenin and hybridization were performed following the Boehringer Mannheim kit instructions.
2.1. Strains and growth conditions C. albicans ATCC 26555 (serotype A) was employed in this study. It was maintained by subculturing every 2^3 weeks on Sabouraud-dextrose agar and propagated in Lee medium [8]. Escherichia coli DH5K was used routinely for transformations. E. coli NM522 was used as host strain for the phage VExCell (Pharmacia). Standard DNA manipulation techniques were carried out as described [9]. 2.2. cDNA library construction and screening A C. albicans cDNA library in phage expression vector VExCell (Pharmacia) was constructed with RNA isolated from protoplasts regenerating for 3 h as yeast cells. The library contained 1.16U104 clones with an average insert size of 1.5 kb. Protoplasts were obtained and regenerated for 1 h as previously described [10]. For mRNA isolation and cDNA synthesis, mRNA Puri¢cation Kit and cDNA Synthesis Kit, both from Pharmacia, were used according to the manufacturer's instructions. cDNAs obtained were subsequently linked to EcoRI-NotI adapters prior to their insertion into EcoRI-digested VExCell. A commercially available rabbit polyclonal antiserum raised against human phosphatidylinositol 3P kinase (PI3Pk) (Upstate Biotechnology Inc.) [11] was used to screen the cDNA library as previously described [12]. Subsequent fragment isolation and subcloning were done according to established protocols.
2.4. Northern and Southern analysis Protoplasts of C. albicans growing at 28³C or 37³C for varying periods were washed and resuspended in 0.1 M LiCl, 0.01 M EDTA, 0.01 M Tris-HCl, pH 7.4, 0.2% (w/v) sodium dodecyl sulfate (SDS) (LETS bu¡er) containing 0.6 M KCl as osmotic stabilizer. Total RNA isolation and Northern blot were carried out essentially as described [12] using disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2P-(5P-chloro)tricyclo[3,3,1,13;7 ]decan}-4-yl) phenyl phosphate (CSPD, Boehringer Mannheim) as a substrate. Genomic DNA isolation and Southern protocols have been described [14]. 2.5. DNA sequencing and analysis DNA sequencing was performed on both strands by the dideoxy chain-termination method [15] with the AutoRead Sequencing Kit (Pharmacia), and cycle sequencing with the fmol Cycle Sequencing System (Promega), following the suppliers' instructions. Sequencing results were analyzed with the ALF DNA Sequencer (Pharmacia Biotech). Nucleotide sequence analysis was performed with the PROSITE Program [16]. Homology searches of NCBI databases were conducted with the BLAST Program [17,18]. The sequence of the CaFRS1 DNA has been deposited in the EMBL database with accession number Y12589.
2.3. Screening of a genomic library
3. Results and discussion
The isolated cDNA fragment was used as a probe to screen a genomic library constructed from C. albicans C9 chromosomal DNA in the plasmid p1041 [13], as per standard procedures [9]. Labelling of
3.1. Cloning of a cDNA encoding phenylalanyl-tRNA synthetase To identify proteins involved in events related to
FEMSLE 8082 19-3-98
182
A. Marcilla et al. / FEMS Microbiology Letters 161 (1998) 179^185
Fig. 2. Alignment of known phenylalanyl-tRNA synthetase L subunit amino acid sequences. The sequences are from C. albicans (CaFRS1), S. cerevisiae cytosolic enzyme (FRS1) [6], C. elegans (Ce Syf) (GenBank accession number Z50044), and E. coli (Ec Syf) (EMBL accession number K02844). Identical residues are boxed.
FEMSLE 8082 19-3-98
A. Marcilla et al. / FEMS Microbiology Letters 161 (1998) 179^185
Fig. 3. Northern blot analysis of the CaFRS1 transcript. Total RNA (20 Wg) from protoplasts grown for 1, 3, 5 and 7 h at 28³C or 37³C were electrophoresed through an agarose gel and transferred to nylon membranes as described in Section 2. The blotted membrane was hybridized with P1 cDNA probe labelled with digoxigenin (A). The corresponding ethidium bromidestained gel is shown in B. The positions of the 28S and 18S ribosomal RNAs are indicated on the left.
the construction of C. albicans cell wall, we immunoscreened a VExCell cDNA library obtained from regenerating protoplasts, which should synthesize de novo an entire cell wall [7,10]. We hypothesized that messengers encoding proteins involved in cell wall regeneration would be highly represented in this library. Fifteen thousand recombinant clones were immunoscreened with a rabbit polyclonal antiserum raised against human PI3Pk [11]. Several positive clones were isolated after three rounds of immunoscreening, and one of them, designated P1, was shown to carry an insert of 1413 bp. Using this cDNA as a probe, a C. albicans genomic library was screened. Nine thousand colonies were screened as described [9], and four positive clones were puri¢ed after three rounds of screening. One of them, designated PA, contained
183
an insert of 7.2 kb. After sequencing an internal fragment of 2028 bp, which contains the entire cDNA clone P1, a single open reading frame of 593 amino acids, extending from base pair 157 up to a stop codon at position 1936, was found (Fig. 1). These data indicated the absence of introns in this gene, and thus we used the cDNA probe for further genomic analysis. A BLAST search in available databases revealed that the nucleotide sequence of PA was highly similar to the Saccharomyces cerevisiae gene FRS1 encoding for the phenylalanyl-tRNA synthetase (PheRS) L (large) subunit [6]. The deduced amino acid sequence of PA showed 63.6% identity with the cytosolic enzyme of S. cerevisiae, and contains the binding site for tRNAPhe , as occurs in S. cerevisiae [19], as well as the potential binding region to the small subunit (K) to form the K2 L2 structure (Fig. 1), required for activity [20]. Fig. 2 shows the alignment of the deduced amino acid sequences of three known PheRS L subunits (SYF) with CaFRS1. The similarities between the S. cerevisiae and C. albicans proteins reached almost 95% in certain regions (amino acids 124^152; 434^ 469 of S. cerevisiae FRS1) [6]. Signi¢cant similarity was observed with the Caenorhabditis elegans deduced amino acid sequence (44.7% identity) (GenBank Sequence Database accession number Z50044). When compared to bacterial enzymes, CaFRS1 exhibited less similarity, dropping to 8.6% identity with the E. coli enzyme (EMBL Sequence Database accession number K02844) (Fig. 2). This similarity was due to several conserved amino acid residues among all enzymes, which would indicate important roles for those residues in the function of the enzymes. An interesting common feature of the deduced amino acid sequence of CaFRS1 with the cytosolic enzyme of S. cerevisiae is the absence of a region in the C-terminus, which is present in bacterial enzymes [21] (positions 698^794 in E. coli) (Fig. 2), in positions 685^780 in the protein of the bacterium Thermus thermophilus [21], and also in S. cerevisiae mitochondrial enzyme [20]. It has been speculated that this extension confers independence from the cytosolic K subunit [21]. The two yeast cytosolic enzymes share a high number of cysteine residues, three being surrounded by similar amino acids and located in
FEMSLE 8082 19-3-98
184
A. Marcilla et al. / FEMS Microbiology Letters 161 (1998) 179^185
identical positions in the predicted amino acid sequence (C28, C266 and C306 of the CaFRS1 sequence) (Fig. 2). Further studies should uncover the function of these regions, but it is tempting to postulate a role in establishing disul¢de bridges needed for the active quaternary structure. 3.2. Southern blot analysis and mapping of the CaFRS1 gene To obtain information about the genomic organization of the CaFRS1 gene, Southern blot analysis was performed using chromosomal DNA digested with EcoRI, and EcoRI plus KpnI and probed with DIG-labelled P1 cDNA. The 1.4-kb probe hybridized only with an 11-kb band when genomic DNA was digested with EcoRI, and two bands of 7 and 4 kb, respectively, when digested with EcoRI plus KpnI, con¢rming the presence of a KpnI site found in the cDNA (data not shown). The probe failed to hybridize with S. cerevisiae genomic DNA digested with EcoRI (data not shown). Although the similarity with FRS1 of S. cerevisiae is high, the differences in the overall DNA sequence and codon usage in the two species could explain this result [22]. These results imply that the CaFRS1 gene is present in a single copy in the genome of C. albicans. Using the same cDNA probe, the chromosomal localization of the CaFRS1 gene was determined. It is located in chromosome R in fragment M of the physical map of C. albicans obtained by fractionation of chromosomes with S¢I [23], being identi¢ed in the fosmids 14E7 and 17D10 (B.B. Magee, personal communication). 3.3. Expression of CaFRS1 reduces during the regeneration of the cell wall in protoplasts To explore transcription of CaFRS1, total RNA was isolated from C. albicans regenerating protoplasts as yeast (28³C) or mycelial (37³C) cells for di¡erent periods, and analyzed by Northern blot hybridization (Fig. 3). A transcript of approximately 1.8 kb was detected, in agreement with the hypothesized size of the gene in C. albicans and FRS1 in S. cerevisiae [6]. The expression of the CaFRS1 transcript decreased during protoplast regeneration at both
28³C and 37³C, being undetectable after 7 h in protoplasts regenerating in the yeast form (Fig. 3). These data could indicate higher stability of the RNA in protoplasts regenerating at 37³C (mycelial cells), although no signi¢cant di¡erences were observed when total RNA from mature cells was analyzed (data not shown). The high level of expression of CaFRS1 in initial steps of protoplast regeneration would suggest a role for phenylalanyl-tRNA synthetase in regulating the synthesis of proteins involved in cell wall synthesis, one of the ¢rst events to occur in protoplasts [10]. Hence, a dual function for tRNA synthetases has been proposed; in addition to their role in tRNA charging, they could regulate protein synthesis, initiating a protein phosphorylation cascade in response to certain stress situations such as temperature changes or deprivation of amino acids [4]. Future studies should analyze the role of tRNA synthetases in processes such as the dimorphic transition originated by stress situations.
Acknowledgments We are grateful to Dr. B.B. Magee (University of Minnesota) for the genomic mapping of the CaFRS1 gene, the generous gift of the C. albicans C9 genomic library, and critical reading of the manuscript. This work was supported by Fondo de Investigaciones Sanitarias de la Seguridad Social (FIS 95/1602), Spain, CICYT (PM96-0019), Spain, and BIOMED2 (BMH4-CT96-0310) Brussels, EU. C.P. was supported by a doctoral fellowship from the Institute of International Cooperation, Spanish Ministry of Foreign A¡airs. M.G.-L. was supported by a doctoral fellowship from CICYT, Spanish Ministry of Culture and Education.
References [1] Odds, F.C. (1988) Candida and Candidosis. A Review and Bibliography. Bailliere Tindal, London. [2] Calderone, R. and Braun, P.C. (1991) Adherence and receptor relationships of Candida albicans. Microbiol Rev. 55, 1^20. [3] Sentandreu, R., Elorza, M.V., Mormeneo, S., Sanjuaèn, R. and Iranzo, M. (1993) Possible roles of mannoproteins in the construction of Candida albicans cell wall. In : Dimorphic Fungi
FEMSLE 8082 19-3-98
A. Marcilla et al. / FEMS Microbiology Letters 161 (1998) 179^185
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11] [12]
[13]
in Biology and Medicine (Van den Bossche, H., Odds, F. and Kerridge, D., Eds.), pp. 169^175. Plenum Press, New York. Clemens, M.J. (1990) Does protein phosphorylation play a role in translational control by eukaryotic aminoacyl-tRNA synthetases? Trends Biochem. Sci. 15, 172^175. Eriani, G., Delarue, M., Poch, O., Ganglo¡, J. and Moras, D. (1990) Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs. Nature 347, 203^206. Sanni, A., Mirande, M., Ebel, J.-P., Boulanger, Y., Waller, J.-P. and Fasiolo, F. (1988) Structure and expression of the genes encoding the K and L subunits of yeast phenylalanyltRNA synthetase. J. Biol. Chem. 263, 15407^15415. Elorza, M.V., Marcilla, A., Sanjuaèn, R., Mormeneo, S. and Sentandreu, R. (1994) Incorporation of speci¢c wall proteins during yeast and mycelial protoplast regeneration in Candida albicans. Arch. Microbiol. 161, 145^151. Lee, K.L., Buckley, M.R. and Campbell, C. (1975) An amino acid liquid synthetic medium for development of mycelial and yeast forms of Candida albicans. Sabouraudia 13, 148^153. 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. Elorza, M.V., Rico, H., Gozalbo, D. and Sentandreu, R. (1983) Cell wall composition and protoplast regeneration in Candida albicans. Antonie van Leeuwenhoek Int. J. Gen. Microbiol. 49, 457^469. Kapeller, R. and Cantley, L. C. (1994) Phosphatidylinositol 3-kinase. BioEssays 16, 565^576. Ramoèn, A.M., Gil, R., Burgal, M., Sentandreu, R. and Valent|èn, E. (1996) A novel cell wall protein speci¢c to the mycelial form of Yarrowia lipolytica. Yeast 12, 1535^1548. Goshorn, A.K., Grindle, S.M. and Scherer, S. (1992) Gene isolation by complementation in Candida albicans and applications to physical and genetic mapping. Infect. Immun. 60, 876^884.
185
[14] Rose, M.D., Winston, F. and Hieter, P. (1990) Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [15] Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463^5467. [16] Bairoch, A. (1993) The PROSITE dictionary of sites and patterns in proteins, its current status. Nucleic Acids Res. 21, 3097^3103. [17] Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403^410. [18] Gish, W. and States, D.J. (1993) Identi¢cation of protein coding regions by database similarity search. Nature Genet. 3, 266^272. [19] Sanni, A., Walter, P., Boulanger, Y., Ebel, J.-P. and Fasiolo, F. (1991) Evolution of aminoacyl-tRNA synthetase quaternary structure and activity: Saccharomyces cerevisiae mitochondrial phenylalanyl-tRNA synthetase. Proc. Natl. Acad. Sci. USA 88, 8387^8391. [20] Aphasizhev, R., Senger, B., Rengers, J.-U., Sprinz, M., Walter, P., Nussbaum, G. and Fasiolo, F. (1996) Conservation in evolution for a small monomeric phenylalanyl-tRNA synthetase of the tRNAPhe recognition nucleotides and initial aminoacylation site. Biochemistry 35, 117^123. [21] Keller, B., Kast, P. and Hennecke, H. (1992) Cloning and sequence analysis of the phenylalanyl-tRNA synthetase genes (pheST) from Thermus thermophilus. FEBS Lett. 301, 83^ 88. [22] Brown, A.J.P, Bertram, G., Feldmann, P.J., Peggie, M.W. and Swoboda, R.K. (1991) Codon utilisation in the pathogenic yeast Candida albicans. Nucleic Acids Res. 19, 4298. [23] Chu, W.-S., Magee, B.B. and Magee, P.T. (1993) Construction of an S¢I macrorestriction map of the Candida albicans genome. J. Bacteriol. 175, 6637^6651.
FEMSLE 8082 19-3-98