Departments of Comparative Genomics1, Discovery Genetics2, Genomics Bioinformatics3, Microbial Bioinformatics and Microbial Genetics4, Glaxo SmithKline, King of Prussia, PA 19406, USA
Author for correspondence: George P. Livi. Tel: +1 610 270 7717. Fax: +1 610 270 7962. e-mail: george_p_livi{at}gsk.com
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ABSTRACT |
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Keywords: aromatic amino acids, GCN4, MET3 promoter, pathogenic fungi
Abbreviations: 3AT, 3-aminotriazole; DAHP, 3-deoxy-D-arabinoheptulosonate 7-phosphate; 5-FOA, 5-fluoroorotic acid; GCRE, Gcn4p-responsive element; SC, synthetic complete; UTR, untranslated region
The GenBank accession number for the sequence reported in this paper is U53216.
a Present address: AstraZeneca Pharmaceuticals LP, 725 Chesterbrook Blvd, Building C-2E08A, Wayne, PA 19087-5677, USA.
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INTRODUCTION |
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Cells of the yeast Saccharomyces cerevisiae contain two isozymes of DAHP synthase encoded by the genes ARO3 and ARO4 (Teshiba et al., 1986 ; Paravicini et al., 1988
, 1989
; Kunzler et al., 1992
). Transcription of both genes is coordinately controlled by Gcn4p, which binds to Gcn4p-responsive elements (GCREs) in the promoters. Enzyme activity is also regulated at the post-translational level, i.e. the activity of Aro3p is feedback-inhibited by phenylalanine, whereas Aro4p is inhibited by tyrosine (Hinnebusch, 1990
; Braus, 1991
). Many prokaryotes possess a third DAHP synthase whose activity is inhibited by tryptophan. Existence of a third isozyme has also been reported in the fungus Neurospora crassa, although the corresponding gene has not been identified (Nimmo & Coggins, 1981
).
We are studying the nature and complexity of DAHP synthases in the pathogenic fungi as potential anti-fungal drug targets, since: (1) humans lack a comparable biosynthetic pathway instead they rely on dietary sources for phenylalanine and tryptophan, and can only synthesize tyrosine via hydroxylation of phenylalanine; (2) based on pathogenicity studies of other auxotrophic mutants of Candida albicans and Cryptococcus neoformans (Manning et al., 1984 ; Shepherd, 1985
; Kirsch & Whitney, 1991
; Perfect et al., 1993
), aromatic amino acid auxotrophs are predicted to display decreased virulence in vivo as a result of poor growth due to suboptimal amino acid bioavailability; and (3) certain amino acid biosynthesis inhibitors have been used safely and effectively as herbicides (reviewed by Kishore & Shah, 1988
).
We previously cloned an ARO3 gene orthologue from the diploid pathogenic fungus C. albicans and found that it can complement an aro3 aro4 double mutation in S. cerevisiae, and that complementation is inhibited by excess phenylalanine (Pereira & Livi, 1993 ). Expression of C. albicans ARO3 mRNA is induced in response to amino acid starvation, consistent with the presence of two putative GCREs in the promoter sequence (Pereira & Livi, 1995
). A homozygous aro3-deletion mutant strain was constructed and found to be prototrophic (Aro+) on synthetic complete media lacking aromatic amino acids (Pereira & Livi, 1996
), suggesting the existence of at least one additional isozyme. A small genomic DNA fragment was PCR-amplified from the mutant strain using degenerate primers, and its nucleotide sequence was found to predict a DAHP-synthase-related peptide with a strong homology to S. cerevisiae Aro4p (Pereira & Livi, 1996
). In this study, we have cloned the complete gene defined by this DNA fragment and determined its evolutionary relationship to known DAHP synthases. We have evaluated its expression in response to nutrient deprivation and the effect of feedback inhibition on its gene product. In addition, we have created strains of C. albicans deficient in Aro3p and Aro4p, and show that they display a conditional growth phenotype in vitro, indicating the presence of only two DAHP synthases.
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METHODS |
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Cloning the ARO4 gene.
Pereira & Livi (1996) previously described using degenerate PCR primers to amplify a 222 bp DNA fragment encoding a DAHP-synthase-related peptide from C. albicans strain SPC64, a homozygous
aro3 mutant. This DNA fragment was labelled and used as a probe to screen a C. albicans strain B792-derived YEp13-based genomic library carried in Escherichia coli (Rosenbluh et al., 1985
). Hybridizations and stringency washes were carried out using the Rapid Hyb system (Amersham Life Sciences) according to the manufacturers protocol.
Phylogenetic analyses.
Publicly available databases, including partial genomic sequences, were searched for homologous ORFs to class I DAHP synthases from S. cerevisiae (Aro3p and Aro4p) and E. coli (AroF, AroG, and AroH) using the programs BLASTP, TBLASTN (Altschul et al., 1990 ) and PSI-BLAST (Altschul et al., 1997
). Individual protein datasets were initially aligned using the program CLUSTAL W v1.7 (Thompson et al., 1994
) with default settings. Multiple sequence alignments were further refined manually using the program SEQLAB of the GCG v10.0 software package (Genetics Computer Group) with reference to the three-dimensional structure of E. coli phenylalanine-regulated AroG (Shumilin et al., 1999
). Regions with residues that could not be unambiguously aligned or that contained insertions or deletions were removed from the alignments. Maximum-parsimony analysis was done using the software package PAUP* (Swofford, 1999
). The number of minimal trees and their lengths were estimated from 100 replicate random heuristic searches. Confidence limits of branch points were estimated by 1000 bootstrap replications. Neighbour-joining analysis was performed using the programs NEIGHBOR and PROTDIST of the PHYLIP 3.57c package (Felsenstein, 1993
). In PROTDIST, the Dayhoff option was invoked, which estimates the expected amino acid replacements per position between all pairs of sequences based on the Dayhoff 120 substitution matrix (Dayhoff et al., 1972
). The programs SEQBOOT and CONSENSE were used to estimate the confidence limits of branching points from 1000 bootstrap replications. The program TREEVIEW (v1.6.1) was used to visualize trees and prepare figures (Page, 1996
).
Plasmid constructions.
The plasmid pMB-7 (constructed by Dr N. Gow and a kind gift from Dr W. Fonzi, Georgetown University, Washington, DC, USA) contains the C. albicans URA3 gene (1365 bp ScaIXbaI fragment; Gillum et al., 1984 ; Losberger & Erst, 1989
) flanked by 1150 bp direct repeat sequences of the Salmonella typhimurium hisG gene (Alani et al., 1987
; Fonzi & Irwin, 1993
). The C. albicans ARO4 disruption plasmid pMB7
aro4 was made by first amplifying a portion of ARO4 from the isolated YEp13-ARO4 library plasmid using primers 5'-TCTTCAGATCTATCACCGATGGATGTGTTTCTTG-3' and 5'-ATTATGAGCTCGATCTTGATGAATTAGAAATTGA-3' (restriction sites italicized), digesting with BglII and SacI and subcloning into the corresponding sites of pMB-7, resulting in plasmid pMB7ARO43'. The subcloned 573 bp PCR fragment of pMB7ARO43' corresponds to nucleotides 1466 to 2038 of the ARO4 gene (see GenBank U53216). Following sequence verification, a second 639 bp PCR product (corresponding to nucleotides 621 to 1259 of the GenBank sequence) was amplified as above using primers 5'-TTACAGCATGCGTAATGATGACAGATGTATTGT-3' and 5'-TCATAGTCGACTTGGATCGGTACATTTGGCA-3' (restriction sites italicized), digested with SphI and SalI and subcloned into the corresponding sites of pMB7ARO43', resulting in pMB7
aro4. This plasmid was digested with SacI and SphI to liberate a 5·2 kb ARO4 disruption cassette in which a 205 bp portion of the ARO4 coding region (nucleotides 1260 to 1465) was replaced by hisG::URA3::hisG. Plasmid pCaDis-aro4 was constructed by PCR-amplifying a portion of the 5' coding region of C. albicans ARO4 (nucleotides 367 to 667 of the GenBank sequence) as above using primers 5'-AAAGGATCCTAGTTATTCTTTGTCAAATAA-3' and 5'-AAACTGCAGGTGAATTGAACATGGACCAAC-3' (restriction sites italicized), digesting with BamHI and PstI, and subcloning the resulting DNA fragment into the corresponding sites in pCaDis [Plasmid pCaDis (Care et al., 1999
) was kindly provided by P. E. Sudbery, University of Sheffield, UK]. pCaDis-ARO4 was linearized with BplI, which cuts the plasmid uniquely within the ARO4 sequence, prior to transformation of C. albicans.
Transformation of S. cerevisiae and C. albicans.
Lithium acetate transformation of S. cerevisiae and C. albicans was performed according to the methods of Gietz et al. (1992) and Sanglard et al. (1996)
, respectively.
Northern-blot analysis.
Total RNA was extracted from C. albicans strain B311-A with and without histidine starvation (described above). Poly(A)+ RNA was prepared by oligo-dT affinity chromatography (mRNA purification kit; Boehringer Mannheim). mRNA was fractionated on a 1·5% denaturing agarose gel in the presence of 2·2 M formaldehyde, transferred to nitrocellulose and probed simultaneously with the 32P-labelled (random primed) 222 bp PCR-generated fragment used to clone the ARO4 gene (see above) and with an approximately 0·8 kb cDNA corresponding to the CYP1 gene encoding cytoplasmic cyclophilin (Koser et al., 1990 ).
Genomic analysis of C. albicans strains.
Strains of C. albicans were analysed using whole-yeast cell PCR (Sathe et al., 1991 ) and by Southern blotting (Southern et al., 1975
). Primers used in ARO4 disruption analysis were 5'-ATTGCCAAATGTACCGATCCAAGT-3' and 5'-CAAGAAACACATCCATCGGTGATA-3', which are designed to amplify the 205 bp deletion/insertion site (see above) in ARO4. PCR analysis of Met3p::ARO4 strains was performed as above using primers 5'-TCCAAGTGTTGTCACTTTCTT-3' (MET3 promoter; nucleotides 5221 to 5242 of pCaDis) and 5'-CATGATAATCACCAAGTCATC-3' (reverse complement of ARO4 nucleotides 728 to 749). PCR screening for the loss of the wild-type ARO4 allele was performed using primers 5'-TCCAAGTGTTGTCACTTTCTT-3' (MET3 promoter; nucleotides 5221 to 5242 of pCaDis) and 5'-ATTTGTTCAGCAACACTGTCT-3' (reverse complement of nucleotides 1330 to 1350 of ARO4, deleted in the aro4
::his3 allele). Southern blotting was performed by digesting C. albicans genomic DNA with BstXI or BstXI and HpaI overnight and separating on a 0·8% agarose gel prior to transfer to nitrocellulose and probing. Probes were generated by PCR and correspond to nucleotides 121 to 1611 and nucleotides 1456 to 1912 of ARO4; they were labelled with 32P using the High Prime DNA Labelling Kit (Boehringer Mannheim Biochemicals).
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RESULTS AND DISCUSSION |
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Database searches confirmed the existence of three DAHP synthases in the proteobacteria E. coli and Salmonella typhimurium. However, most complete genome sequences of bacteria as well as fungi reveal fewer than three types of DAHP synthase. For example, the proteobacterium Haemophilus influenzae has only AroG. The sequenced genomes of three fungal species reveal AroG and AroF but not AroH. Thus, the evolution of the different feedback-inhibited types of DAHP synthases appears to be highly species specific.
Generally consistent phylogenetic trees were generated by the maximum-parsimony (MP) and neighbour-joining (NJ) methods. In 100 heuristic searches, MP detected only one shortest tree, which was 2158 steps in length. Phylogenetic analyses show four distinct clusters of DAHP synthases (Fig. 2). The yeast DAHP synthases form one cluster, which is highly divergent from bacterial enzymes. Within the fungal clade, S. cerevisiae and C. albicans Aro3p (AroG-type) and Aro4p (AroF-type) DAHP synthases occurred in clusters specific to each amino acid inhibitor. Although Sch. pombe Aro3p and Aro4p appear to be ancestral to other fungal DAHP synthases, this might be an artifact of their more rapid sequence evolution. Among the bacteria, the three different feedback-inhibited enzymes, AroF, AroG and AroH, formed separate clades. AroG was split into two groups: one consisted of proteobacteria and the other of high-G+C Gram-positive bacteria (Mycobacterium avium) and actinobacteria (Corynebacterium glutamicum and Amycolatopsis methanolica). While bootstrap and minimal tree search support for five separate clades of DAHP synthases was generally high, the resolution of branching order among the groups was not resolved. NJ provided the best support for clustering the yeast enzymes with bacterial AroG although the bootstrap value (47%) was less than 50%. Regardless, phylogenetic analysis suggests that the two different fungal DAHP synthases arose from an early gene duplication in the fungi.
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Functional expression in S. cerevisiae
The C. albicans ARO4 gene carried on YEp13 was introduced into S. cerevisiae strain RH1368 (aro3-2 aro4-1 gcn1-1 trp1-1 leu2-2), selecting for Leu+ transformants. The gcn1-1 mutation renders the cells Gcn4p-deficient, so that functional complementation should correspond to basal gene expression (Hinnebusch, 1990 ). As shown in Fig. 4(a
, b
), the C. albicans ARO4 gene, like ARO3 (Pereira & Livi, 1993
), complements the aro3 aro4 mutations in S. cerevisiae. The ARO3 gene from C. albicans was previously defined by its inability to complement when cells are grown on excess (2 mM) phenylalanine, presumably due to isozyme-specific feedback inhibition (Pereira & Livi, 1993
). Complementation by ARO4 was similarly impaired (albeit to a lesser degree) by excess (5 mM) tyrosine (Fig. 4c
), but not excess (5 mM) phenylalanine or tryptophan (Fig. 4c
and data not shown). Taken together, these data suggest that C. albicans Aro4p is a structural as well as functional orthologue of S. cerevisiae Aro4p.
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To address this possibility, three alternative strategies were tried. First, strain SPC311 was transformed with plasmid DNA in which the orientation of the hisG-URA3-hisG cassette was reversed relative to the flanking ARO4 sequences in plasmid pMB7aro4, with the intent to reduce the occurrence of double crossover events between the hisG repeats of the introduced DNA and the hisG of the previously disrupted allele. The second was a nested PCR-based disruption strategy (Wilson et al., 1999
) in which the URA3 marker was amplified using primers containing 90 nucleotides of flanking ARO4 homology. The third strategy was to use the AUR1 dominant selectable marker (Hashida-Okado et al., 1998
) to disrupt the final copy of ARO4. None of these strategies produced a strain in which the second ARO4 allele was disrupted. In addition, the medium used to select for double aro4 disruptants in a
aro3::hisG/
aro3::hisG background was supplemented with increased concentrations of each of the three aromatic amino acids in an attempt to rescue the potential auxotrophy of the double mutant. Despite these efforts we were unable to generate a disruption of the remaining wild-type copy of ARO4 in strain SPC311. Similarly, we were unable to disrupt the remaining wild-type ARO3 allele in strain SSC9 (Table 2
) using the
aro3::hisG-URA3-hisG deletion/insertion cassette previously described by Pereira & Livi (1996)
(data not shown).
Construction of an inducible double aro3 aro4 knockout strain
Recently, a system has been developed that utilizes the promoter of the tightly regulated MET3 gene of C. albicans to assess essentiality and null phenotype of genes in this organism (Care et al., 1999 ; Warit et al., 2000
). The product of the C. albicans MET3 gene is required for the biosynthesis of cysteine and methionine and its expression is strongly repressed in the presence of these amino acids (Care et al., 1999
). We constructed an integrating plasmid (pCaDis-Aro4) in which an extreme 5' fragment of the ARO4 coding region was placed immediately 3' of the C. albicans MET3 promoter. Transformation with this plasmid after restriction with BplI within the ARO4 sequence results in a duplicative integration in the genome resulting in a 3' truncated version of the ARO4 gene as well as a full-length copy under the control of the MET3 promoter (see schematic in Fig. 7a
).
We started with strain SPC311 (aro3::hisG/
aro3::hisG
aro4::hisG/ARO4), created using the Ura-blaster technique (as described above). Linearized plasmid pCaDis-Aro4 was introduced into cells of SPC311 and Ura+ transformants were selected. Transformants were screened by genomic PCR to identify those containing plasmid integrations at the remaining ARO4 locus. A strain with the correct genotype was identified and called SSC12 (
aro3::hisG/
aro3::hisG
aro4::hisG/Met3p::ARO4). Proper integration was verified by Southern blotting (Fig. 7b
, lane 4). A second isolate with identical genotype was also identified (strain SSC13, Fig. 7b
, lane 5).
This strain was tested for growth in the presence or absence of aromatic amino acids under conditions of ARO4 expression or repression (Fig. 8). In the absence of cysteine and methionine and the aromatic amino acids, strain SSC12 grows equally as well as its isogenic parent SPC208, and its progenitor strain CAI4, presumably as a consequence of ARO4 expression. In contrast, strain SSC12 fails to grow in media supplemented with 2·5 mM cysteine and methionine in the absence of the aromatic amino acids tryptophan, tyrosine and phenylalanine. Thus, switching off expression of the last remaining copy of ARO4 results in a conditional growth defect, and both Aro3p and Aro4p are apparently necessary for the growth of C. albicans on synthetic complete medium in the absence of exogenously supplied aromatic amino acids (Fig. 8
). Inclusion of the three aromatic amino acids at standard concentrations (0·2 mM each) did not restore growth to wild-type ARO4 levels (e.g. CAI4 and SPC208), but under these conditions some slow growth of SSC12 was observed (data not shown). Slow growth was also observed on rich (YEPD) medium, presumably due to the presence of some inhibitory levels of cysteine and methionine, and was inhibited further with the addition of 2·5 mM each of cysteine and methionine (data not shown).
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In the present study we have demonstrated that cells of C. albicans contain a second, Aro4p-related, DAHP synthase whose expression is coordinately controlled by a GCN-like mechanism and whose activity is sensitive to feedback inhibition by tyrosine. In addition we have shown that in C. albicans, as in S. cerevisiae, Aro3p and Aro4p represent the only functional isozymes of DAHP synthase present in the cell. In contrast to studies in S. cerevisiae, however, we have found that when these isozymes are absent, cells growth is severely inhibited even when supplemented with phenylalanine, tyrosine and tryptophan. These results may partially explain our inability to generate a true aro3/aro3
aro4
/aro4
C. albicans mutant. Cells devoid of DAHP synthase activity are highly compromised in terms of growth in vitro, but the observed growth defect can be restored to some extent by excess aromatic amino acids as well as certain metabolic intermediates of the aromatic amino acid pathway. The question remains, however, whether this pathway offers tractable targets for development of antifungal drugs. Although it has proven difficult thus far, we are continuing to try to create a true aro3
/aro3
aro4
/aro4
mutant under appropriately supplemented growth conditions for use in pathogenicity models for further target validation.
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ACKNOWLEDGEMENTS |
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Received 9 November 2001;
revised 4 December 2001;
accepted 7 January 2002.
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