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Citrate synthase is a central activity in carbon metabolism. It is required for the tricarboxylic acid (TCA) cycle, respiration, and the glyoxylate cycle. In Saccharomyces cerevisiae and Arabidopsis thaliana, there are mitochondrial and peroxisomal isoforms encoded by separate genes, while in Aspergillus nidulans, a single gene, citA, encodes a protein with predicted mitochondrial and peroxisomal targeting sequences (PTS). Deletion of citA results in poor growth on glucose but not on derepressing carbon sources, including those requiring the glyoxylate cycle. Growth on glucose is restored by a mutation in the creA carbon catabolite repressor gene. Methylcitrate synthase, required for propionyl-coenzyme A (CoA) metabolism, has previously been shown to have citrate synthase activity. We have been unable to construct the mcsAΔ citAΔ double mutant, and the expression of mcsA is subject to CreA-mediated carbon repression. Therefore, McsA can substitute for the loss of CitA activity. Deletion of citA does not affect conidiation or sexual development but results in delayed conidial germination as well as a complete loss of ascospores in fruiting bodies, which can be attributed to loss of meiosis. These defects are suppressed by the creA204 mutation, indicating that McsA activity can substitute for the loss of CitA. A mutation of the putative PTS1-encoding sequence in citA had no effect on carbon source utilization or development but did result in slower colony extension arising from single conidia or ascospores. CitA-green fluorescent protein (GFP) studies showed mitochondrial localization in conidia, ascospores, and hyphae. Peroxisomal localization was not detected. However, a very low and variable detection of punctate GFP fluorescence was sometimes observed in conidia germinated for 5 h when the mitochondrial targeting sequence was deleted.
There has been increased interest in primary carbon metabolism in fungi in recent years. There are two main reasons for this. As fungal pathogens establish infection they must adapt their utilization of carbon sources to the substrates present in the new environment of the host cells (reviewed in reference 6). With many of the fungal genomes available, the number of genes encoding enzymes and transporters potentially involved in central metabolism has become apparent and is greater than might have been anticipated (for example, see reference 16). Deciphering this complexity requires not only genome-wide studies but also detailed studies of individual genes encoding these proteins in order to determine their regulation and the cellular localization of the proteins, as well as their roles in metabolism and development. Here we report molecular genetic analysis of the citA gene encoding citrate synthase (EC 188.8.131.52), a central enzyme of carbon metabolism, in the filamentous ascomycete Aspergillus nidulans.
Citrate synthase is required for the formation of citrate from acetyl-coenzyme A (CoA) and oxaloacetate in the tricarboxylic acid (TCA) cycle and is therefore necessary for respiratory growth as well as for the generation of intermediates for biosynthetic reactions. Together with aconitase, malate dehydrogenase, isocitrate lyase, and malate synthase, it is also an essential enzyme in the glyoxylate cycle, which is necessary for growth on carbon sources such as acetate, ethanol, and fatty acids which are catabolized via acetyl-CoA (reviewed in reference 26).
In Saccharomyces cerevisiae the mitochondrial Cit1 is the major citrate synthase of the TCA cycle. An additional enzyme, Cit2, is peroxisomally localized via a C-terminal peroxisomal targeting sequence (PTS1) (29). In response to mitochondrial dysfunction CIT2 is upregulated via the retrograde response mediated by RTG1, -2, and -3, while mitochondrial respiratory deficiency results in RTG-dependent expression of CIT1 as well as that of aconitase (ACO1) and isocitrate dehydrogenase (IDH1 and IDH2), all enzymes necessary for 2-oxoglutarate formation and hence the synthesis of glutamate required for amino acid biosynthesis (9, 15, 30). In addition a third gene, CIT3, encodes a mitochondrial enzyme with citrate synthase activity. This enzyme has greater activity with propionyl-CoA, forming methylcitrate, and is necessary for the mitochondrial methylcitrate cycle involved in the metabolism of propionate (24). Cit2 has also been proposed to have methylcitrate synthase activity (17).
In S. cerevisiae Cit2 also plays a role in the transfer of acetyl-CoA generated in peroxisomes by β-oxidation of fatty acids or by ethanol and acetate metabolism in the cytoplasm to the mitochondria for metabolism via the TCA cycle. There are two alternative pathways: transfer as acetyl-carnitine formed by the peroxisomal/mitochondrial carnitine acetyltransferase Cat2, together with the cytoplasmic Yat1 and Yat2 carnitine acetyltransferases, or transfer via citrate formed by Cit2 (45, 51, 52). Only disruption of both pathways (e.g., by deletion of CAT2 and CIT2) results in a growth defect on fatty acids. The fact that deletion of CIT2 is not essential for utilization of carbon sources metabolized via acetyl-CoA indicates that mitochondrial citrate synthase activity can replace the peroxisomal activity in the glyoxylate cycle. In contrast, in the pathogenic yeast Candida albicans, there is a single gene for citrate synthase and it is mitochondrial, and acetyl-CoA transport to mitochondria is solely dependent on the carnitine pathway (43, 57). In the plant Arabidopsis thaliana, there are five genes encoding citrate synthase enzymes. Two are peroxisomal (CSY2 and CSY3) and required for fatty acid respiration and seed germination, indicating that carnitine acetyltransferases are not required for shuttling acetyl units to the mitochondria (37).
The filamentous ascomycete Aspergillus nidulans has both citrate synthase-encoding and methylcitrate synthase-encoding genes, citA and mcsA, respectively (8, 36). In both A. nidulans and Aspergillus fumigatus it has been shown that McsA is mitochondrial and has both methylcitrate and citrate synthase activities and is required for propionyl-CoA metabolism (8, 22, 31). Cell fractionation studies have shown that citrate synthase activity colocalizes with the mitochondrial fraction (35), and an N-terminal mitochondrial targeting sequence is predicted by the gene sequence (36). However, CitA has a putative C-terminal peroxisomal targeting sequence (PTS1 AKL), and genes in some filamentous ascomycetes also have potential PTS1 sequences (see below). The role of peroxisomal citrate synthase activity is not at all clear. The acuJ-encoded peroxisomal/mitochondrial carnitine acetyltransferase is required for growth on both fatty acids and acetate, while the facC-encoded cytoplasmic enzyme is required for growth on acetate (1, 20, 42). Therefore, like C. albicans, the carnitine shuttle is absolutely required for acetyl-CoA intracellular transport.
Because of our interest in the role of peroxisomes in fatty acid and acetate metabolism in A. nidulans (21), we have investigated phenotypes resulting from deletion of the citA gene. Our results indicate that loss of CreA-mediated carbon repression allows expression of mcsA, resulting in the restoration of sufficient citrate synthase activity to suppress growth and developmental defects resulting from citAΔ. We have also investigated the role of peroxisomal localization of CitA and found this is at most extremely low and does not play a major role.
Media and conditions for growth of A. nidulans were as described previously (21). All strains were derived from the original Glasgow strain and contained the velA1 mutation, and standard A. nidulans genetic manipulations were as previously described (48, 49). Preparation of protoplasts and transformation were as described previously (32). Recipient strains contained nkuAΔ to promote homologous integration events, and selectable markers were the bar gene (glufosinate resistance) and riboB+ from A. fumigatus (32). A strain containing mcsAΔ (RYQ11-56) was obtained from Matthias Brock. DNA from transformants was analyzed by Southern blotting to confirm predicted integration events. Standard methods for DNA manipulations, RNA isolation, nucleic acid blotting, and hybridization have been described previously (20, 39).
Aspergillus sequences were obtained from the genome sequences available from the Broad Institute (http://www.broadinstitute.org/annotation/genome/aspergillus_group/MultiHome.html). Other fungal sequences were derived from either specific genome sequences available at the Broad Institute (http://www.broadinstitute.org/annotation/fungi/index.html) or at NCBI (http://www.ncbi.nlm.nih.gov/).
The citA gene (coordinates −910 to +2570) was amplified using the primers 5′-GGGCTTGACGGTAATTTGAA and 5′-AGTTGGGACGACAACAGTCC and cloned into pBluescript SK(+) (Stratagene, La Jolla, CA), forming pSM7337. A deletion construct was made by replacing a partial XhoI-partial PstI fragment, corresponding to amino acids +56 to 420 of the predicted protein, with a partial XhoI-partial PstI fragment of the bar selectable marker, creating pSM7341. A linear PCR product generated using the above primers was transformed into strain TNO2A21 (32), and transformants were selected for glufosinate resistance.
The plasmid pRG6501 contains from +812 to +2966 of the wA coding region (amplified using the primers 5′-TATGGTGCCAATCCACGG and 5′-TGATGGAAGATCCTGGCC) and was cloned into a plasmid containing the A. fumigatus riboB gene inserted into the SspI site of pBluescript SK(+). Upon transformation into a recipient wA+ A. nidulans strain, insertion of the circular plasmid by homologous recombination causes a disruption of the wA gene, which can be detected by the presence of white conidia.
A fragment containing the citA gene (coordinates −910 to +2570) was cloned into pRG6501 for targeting to the wA locus. This was transformed into the citAΔ strain grown overnight in 0.05% glucose plus 50 mM acetate liquid medium, and a transformant with integration of a single plasmid copy at wA was obtained (citA+/citAΔ). A transformant with pRG6501 integrated at wA was obtained as a control.
For citAK468* the primers 5′-CTAGGCGTAAGCCTCAGTGC and 5′-CTTGTTGGCGCTAAGCTATA were used to change A 1829 to T, which resulted in the amino acid change from K468 to a stop codon. For citAAK* the primers 5′-CTTAGCGCCAACAAGCTTGG and 5′-TAAATTTCTTGAACGACTGAGG were designed to delete the codon +1847 to +1849 (L474). Inverse PCR using Phusion (Finnzymes) and these primers was performed on the plasmid pSM7340 containing +1173 to +2326 of the citA gene. The resulting product was ligated to form the plasmids pSM7344 and pSM7419 for citAK468* and citAAK*, respectively. Full-length versions of the constructs were reconstructed by replacing the 624-bp PstI-EcoRI fragment from the citA+ gene with that from pSM7344 and pSM7419, and these full-length versions were cloned into the wA-targeting plasmid pRG6501 and transformed into the citAΔ strain to generate citAAK*/citAΔ and into TNO2A21 to generate citAK468*/citA+.
For localization studies, a fragment encoding AcGFP (Clontech Laboratories, Inc.) was used to replace bp +823 to +972, corresponding to amino acids +174 to +226 of citA and citAAK*, forming citA-gfp and citAAK*-gfp, respectively. These were inserted into the wA-targeting vector and transformed into TNO2A21, generating citA-gfp/citA+ and citAAK*-gfp/citA+.
For citA3-34Δ the primers 5′-GCCATATCTGATCAAACAAC and 5′-CTCTACCGGCAAGACCAAG were designed to delete bp +6 to +101, corresponding to amino acids 3 to 34 of citA. Inverse PCR was performed with pSM7337 as template using Phusion (Finnzymes), and the product was ligated to form pSM7480. The 1.1-kb XbaI-XhoI fragment from pSM7480 containing the deletion was used to replace the corresponding region in both the citA+and citA-gfp sequences in the wA-targeting plasmids for transformation into TNO2A21 to generate citA3-34Δ/citA+ and citA3-34Δ-gfp/citA+.
RNA extractions and reverse transcriptase PCRs (RT-PCRs) were carried out as described previously (55). The annealing temperature for all primer sets was 58°C. citA was amplified with the primers 5′-ACCGCAATGTTTTCAAGGAC and 5′-GCGTTGAGGGTAGACCAGAG to yield a cDNA (364 bp) using 24 cycles. mcsA was amplified with the primers 5′-ATCCTAGCGATGCAGGAGAA and 5′-GGTAGAAGAGCACGCCTGAG to yield a cDNA (300 bp) using 28 cycles. benA (loading control) was amplified with the primers 5′-AGTTGTTACCAGCACCGGAC and 5′-GCTCCGGTGTTTACAATGG to yield a cDNA (209 bp) using 26 or 25 cycles.
Conidia were grown in 24-well plates containing 1% glucose minimal liquid medium or minimal medium containing 50 mM acetate at 37°C. Photos were taken at the indicated time points by using an inverted microscope, and germinated conidia were counted (detectable germ tube emergence). At least three replicates were performed, and a minimum of 50 conidia were counted for each replicate, but most often there were more than 100.
For hyphal samples, strains were grown for 16 h at 37°C in 1% glucose minimal liquid medium and transferred for 4 h to either the same medium or minimal medium including 50 mM acetate and 10 mM NH4Cl. For germinated conidia, strains were grown for 5 h at 37°C in 1% glucose minimal liquid medium. Total protein extraction and Western blotting were performed as previously described (50). Aliquots of 100 μg of total protein for both hyphal and conidial germination samples were separated by SDS-PAGE. CitA-GFP was detected using a 1/5,000 dilution of anti-GFP rabbit polyclonal antibody (Millipore) and a 1/4,000 dilution of anti-rabbit IgG–horseradish peroxidase antibodies (Promega) as primary and secondary antibodies, respectively. Signals were detected using a Fujifilm image reader LAS-3000 (Berthoid Australia Pty., Ltd.). Tubulin was detected as previously described (55).
For hyphae, strains were grown on coverslips for 16 h at 37°C in 1% glucose minimal liquid medium and then transferred to the same medium, 50 mM acetate, or 0.5% Tween 80 with ammonium chloride as a nitrogen source for a further 4 h. Strains were incubated for 1 h with 5 μΜ MitoTracker Red CMXRos (Invitrogen) before fixing as previously described (46). Confocal microscopy was performed on hyphae, conidia, and ascospores by using an inverted Olympus FV1000 twin-scanning confocal microscope (Olympus Australia) with a 100× oil objective. For germinated conidia, strains were grown on coverslips in 1% glucose minimal liquid medium at 37°C for 5 h. Images were captured using an Olympus BX60 confocal microscope and Olympus DP71 camera. Images of cleistothecia were captured on an Olympus SZX12 microscope.
The structural gene for citrate synthase (citA) of A. nidulans has previously been cloned and sequenced (36). This corresponds to AN8275 in the genome sequence (http://www.broadinstitute.org/annotation/genome/aspergillus_group/MultiHome.html). A 3.4-kb fragment containing this gene was cloned, and a deletion construct was made by replacing sequences with coordinates (relative to the ATG) +235 to +1684 (corresponding to amino acids 56 to 420) with the bar gene encoding glufosinate resistance. A 4-kb linear fragment generated by PCR was transformed into an nkuAΔ strain, and transformants were selected for glufosinate resistance on protoplast medium, which contains 1 M sucrose and 1% glucose. The few large resistant colonies appearing on the plates were subsequently found by Southern blot analysis to be due to integration events without generating a gene deletion. A longer incubation period yielded small resistant colonies which were found to grow more strongly on glufosinate medium when acetate was the sole carbon source. Southern blot analysis of three of these transformants showed that the predicted gene replacement event had occurred, and one was used in subsequent analyses. The citAΔ mutant phenotype on glucose medium was very slow growth. Extended incubation showed that conidiation was not greatly affected (Fig. 1A). All citAΔ phenotypes were complemented by targeting the 3.4-kb citA+ fragment (containing 910 bp of the 5′-untranslated region [UTR] and 718 bp of the 3′-UTR) to the wA locus (Fig. 1A and B). Integration of the empty wA-targeting vector did not restore normal growth on glucose (Fig. 1A).
The small colony phenotype of citAΔ was observed on both minimal medium and on rich complete medium, which contains 1% glucose. In contrast, growth on gluconeogenic carbon sources, including amino acids, acetate, and fatty acids (butyrate and oleate), was only slightly less than the wild type, with the exception of ethanol, on which growth was extremely poor (Fig. 1B). Growth of citAΔ was also restored when glycerol, fructose, lactose, or arabinose, but not xylose, was provided as sole carbon source (Fig. 1C). This pattern of growth correlated with glucose and xylose as strong sources of carbon catabolite repression, as previously described (2).
The creA gene mediates carbon catabolite repression, and the creA204 mutation, resulting from a mutation affecting the DNA binding domain of the CreA repressor, results in relief of repression (40). Therefore, a citAΔ creA204 strain was constructed by crossing and tested for growth on various carbon sources (Fig. 1C). Although the creA204 mutation results in a more compact colony, it was clear that relief of carbon catabolite repression substantially restored growth on the repressing carbon sources glucose and xylose. No effect was seen for growth on ethanol.
In both A. nidulans and A. fumigatus it has been shown that the product of the mcsA gene has both methylcitrate synthase and citrate synthase activities, and the expression of this gene is increased on nonrepressing carbon sources (8, 22, 31). This strongly suggested that derepression of mcsA expression by growth on nonrepressing carbon sources or by the creA204 mutation results in suppression of the effects of loss of citrate synthase activity. mcsAΔ does not affect growth on carbon sources other than leading to propionate sensitivity due to the requirement for methylcitrate synthase in propionyl-CoA metabolism (Fig. 1D) (7, 8). Interestingly, citAΔ resulted in increased utilization and greater resistance to propionate than the wild type (Fig. 1D), indicating that loss of CitA, which has no methylcitrate synthase activity (8), may allow more efficient metabolism of propionyl-CoA via McsA.
It was found that a mcsAΔ citAΔ double mutant strain could not be isolated by crossing. The mcsA gene is closely linked to the yA locus on linkage group I. Plating ascospores from a cross between a yA2 citAΔ strain and a mcsAΔ strain on acetate plus glufosinate medium to select for citAΔ progeny yielded only 1 green conidiating recombinant in 100 progeny, while plating on acetate or proline medium yielded equal numbers of green and yellow conidiating colonies. More detailed scoring of progeny plated on acetate medium from this cross showed that 52 yA2 progeny all had the citAΔ phenotype of poor growth on glucose and propionate resistance, while 50 yA+ progeny were citA+ and propionate sensitive. This is consistent with citAΔ mcsAΔ double mutants being inviable and therefore strongly suggests that McsA provides the alternative citrate synthase activity on carbon-derepressing medium and that this enzyme activity is essential, at least for colony formation from ascospores. Semiquantitative RT-PCR analysis provided further support for this hypothesis (Fig. 1E). Growth on derepressing carbon sources resulted in greater mcsA expression than on glucose. In addition the creA204 mutation resulted in elevated mcsA expression on glucose, in keeping with suppression of citAΔ phenotypes.
A third A. nidulans protein predicted to contain domains characteristic of citrate synthases has been predicted (AN1079). Our data suggest that this protein cannot compensate for the loss of CitA and McsA for the phenotypes we investigated. Furthermore, this protein lacks a mitochondrial targeting sequence and has no obvious peroxisomal targeting sequence. Therefore, the function of this protein remains unknown.
Strains containing citAΔ are able to grow on carbon sources metabolized via acetyl-CoA and which therefore require the glyoxylate cycle. Since McsA can substitute for CitA, as described above, and this activity is mitochondrial, a functional glyoxylate cycle does not depend on cytoplasmic or peroxisomal citrate synthase activity. Nevertheless CitA has a potential C-terminal peroxisomal targeting sequence (AKL), and this is conserved in other Aspergillus spp. and in Penicillium chrysogenum. The C-terminal sequences of some ascomycetes are shown in Fig. 2A. PTS1 sequences are not found in the citrate synthases of other ascomycetes, apart from the defined peroxisomal Cit2 of S. cerevisiae, although the situation for Podospora anserina and Neurospora crassa is ambiguous, because KKL has the potential to act as a PTS1 in the appropriate context (4). The methylcitrate synthases McsA and Cit3 lack PTS1 sequences, and Candida albicans lacks an ortholog of Cit3. A mutation (citAAK*) changing the C-terminal L to a stop codon and therefore altering the putative PTS1 was able to fully complement citAΔ for all growth phenotypes (Fig. 2B). Furthermore, no effect was seen on the utilization of fatty acids or acetate, which require enzyme activities located in peroxisomes (21).
As noted above, no effects of citAΔ on conidiation on any medium were observed. However, sexual spore development was affected. A. nidulans is homothallic, and therefore strains can be selfed. In addition, each fruiting body (cleistothecium) produces ascospores arising from a single initial diploid zygote undergoing sequential meiotic divisions (48). Crosses are normally set up by initially growing strains on glucose-containing complete medium and transferring to glucose minimal medium plates which are taped to exclude air (48). Because citAΔ results in poor growth on glucose, we set up crosses on media with either acetate or proline as the carbon source before transferring to glucose minimal medium plates. It was found that sexual development was initiated in citAΔ selfed crosses. However, cleistothecia were small and obscured by the nurse cells surrounding the cleistothecia (Hulle cells), and this was also shown by comparing sizes of cleaned isolated cleistothecia (Fig. 3A and B). Measurement of sizes showed that cleistothecia resulting from selfing of citAΔ strains were approximately 50% of the size of those from citA+ (Fig. 3C). In citAΔ × citA+ heterozygous crosses a bimodal size distribution was observed, indicating that the citAΔ phenotype is autonomous (Fig. 3C). This was found to correlate with a complete lack of ascospores in small cleistothecia, although the cleistothecial wall was hard and melanized and the red pigment, cleistothecin, was produced (Fig. 3D). In a heterozygous cross, 9 cleistothecia lacking ascospores had an average size of 124 ± 8 μm, while 25 fertile cleistothecia had an average size of 255± 12 μm. These results are consistent with CitA being required for meiosis but not for development of sexual tissue.
Sexual development and viable ascospore production was not affected in selfings of creA204 citAΔ strains (Fig. 3D). This result indicated that McsA activity in meiotic cells is subject to CreA-mediated repression and that, if citrate synthase activity is required, McsA can substitute for CitA in the growth and development of cells in sexual reproductive tissue but not in meiosis within cleistothecia. In crosses set up on derepressing carbon sources (arabinose and lactose), fertile cleistothecia were produced in a citA+ strain but not in a citAΔ strain. Therefore, the effects of CreA on mcsA repression are intrinsic to meiotic cells and not dependent on the external carbon source.
A mutant version of citA, in which the sequence encoding the residue K468 of CitA was altered to a stop codon (Fig. 2A), was found to be unable to complement citAΔ for growth on glucose medium, indicating that this mutation eliminates enzyme activity. Since this mutation only eliminates the last seven amino acids, it is highly likely to produce an inactive protein. This remains to be confirmed. This mutation also resulted in sterile cleistothecia. This provides some support for the conclusion that the loss of citrate synthase activity rather than loss of the protein itself is responsible for the sexual phenotype. The citAAK* mutation did not affect sexual development, with normal-sized fertile cleistothecia produced in selfings (Fig. 3A and B). Therefore, the possible peroxisomal localization of CitA is not required for fertility.
Germination of citAΔ conidia in glucose medium was delayed by at least 8 h in comparison with the citA+-complemented strain (Fig. 3E). This was partially suppressed by the creA204 mutation, suggesting that citrate synthase activity present in conidia is required for normal rates of germination and that CreA repression of mcsA expression during conidial formation and/or during germination occurs. Germination in acetate medium was slower than in glucose, and at 32 h only 60 to 70% of wild-type conidia were germinated (Fig. 3E). Again, germination of citAΔ conidia was delayed. Germination of the citAΔ creA204 strain in acetate medium was extremely poor for unknown reasons. Germination of the citAAK*/citAΔ strain was similar to that of the citA+/citAΔ strain, showing that peroxisomal localization is not required. As shown below, GFP-tagged CitA was detected in ungerminated conidia.
Although the growth of colonies arising from the inoculation of mass conidia of the citAAK*/citAΔ strain was not detectably affected at 2 to 3 days of incubation (Fig. 2B), it was observed that colonies arising from single ascospores or conidia developed more slowly than citA+ strains, with colonies barely visible at approximately 24 h (Fig. 4A, B, and C). This suggested a possible role for the peroxisomal localization of CitA during the early stages of growth. Colony development from single conidia and ascospores was also investigated in pex mutants, in which peroxisomal localization of matrix proteins is affected (21). Deletion of the pexE gene, encoding the receptor required for the localization of PTS1-containing proteins but not PTS2 proteins (21), resulted in slow colony growth from both conidia and ascospores (Fig. 4E). In contrast, the complete loss of peroxisomes resulting from deletion of pexC or the loss of targeting of all matrix proteins resulting from mutation of pexF did not result in greatly slowed colony development (Fig. 4E). It should be noted that pex mutants show delayed conidial germination, but at 5 to 6 h the germination percentages are similar to the wild type (G. S. Khew, unpublished data). Colony growth arising from single conidia or ascospores of loss-of-function mutants affected in the glyoxylate cycle (acuD, for isocitrate lyase, and acuE, for malate synthase) or gluconeogenesis (acuF, for phosphoenol pyruvate carboxykinase) was not significantly different from wild type (results not shown). Therefore, an essential role for peroxisomal CitA in the utilization of gluconeogenic carbon sources during the early stages of growth following conidial germination is unlikely.
CitA was tagged by inserting a sequence encoding GFP (between coordinates +823 and +972, corresponding to amino acids +174 to +226), and the resulting plasmid was targeted to the wA locus. In hyphae, GFP fluorescence colocalized with mitochondria stained with Mito Tracker Red (Fig. 5A). Fluorescence was not detected in separate organelles that might correspond to peroxisomes. This was particularly clear for oleate-grown hyphae, where we have previously shown that peroxisomes proliferate, forming large clusters (21). Furthermore, we have been able to detect dual localization to both mitochondria and peroxisomes with other GFP-tagged proteins (reference 46 and unpublished data). Therefore, under these conditions, either there is no peroxisomal localization or it is not detectable due to peroxisomes being obscured by mitochondria combined with a low level of expression of the peroxisome-localized form of the enzyme. Western blotting showed expression of CitA-GFP, and this was 2- to 3-fold higher in acetate-grown than in glucose-grown hyphae (Fig. 5E). Expression was not affected by the citAAK* mutation. Both resting conidia and ascospores showed apparent mitochondrial localization of CitA-GFP (Fig. 5B and C). MitoTracker Red staining was not possible, presumably due to mitochondria being inactive (47). The pattern of localization was not affected by mutation of the putative PTS1.
To increase the possibility of detecting peroxisomal targeting we deleted sequences for the predicted mitochondrial targeting sequence (amino acids 3 to 34) in both citA+ and citA-gfp and targeted the mutant genes to the wA locus in a citA+ background. Not surprisingly, isolates of genotype citA3-34/citAΔ, obtained by crossing to a citAΔ strain, were unable to grow on glucose medium, indicating that mitochondrial localization of CitA is essential. CitA3-34-GFP fluorescence was not detectable in hyphae or resting conidia and ascospores (Fig. 5A to C), and no expression was detected by Western blotting (Fig. 5E). Therefore, loss of mitochondrial targeting may result in protein instability.
As noted above we observed slower colony development resulting from the citAAK* mutation. Therefore, we investigated conidia germinated for 5 h, when polarized growth is evident. Western blotting detected high levels of CitA-GFP in both citA+-gfp and citAAK*-gfp strains, and also a low level of expression was clearly detected in the citA3-34Δ-gfp strain, unlike in hyphae (Fig. 5E). GFP fluorescence apparently localized to mitochondria was observed in citA+-gfp germinated conidia. In the absence of the mitochondrial targeting sequence in the citA3-34Δ-gfp strain, punctate fluorescence was observed in a minority of germinated conidia and not in every experiment. Some examples are shown in Fig. 5D. This suggested some peroxisomal localization of CitA at this stage of growth, which was observable in the absence of highly fluorescent mitochondria in citA+-gfp germinated conidia. Support for a requirement for peroxisomal localization of CitA for colony extension was provided by the finding that the citA3-34Δ mutation could complement the citAAK* mutation for colony extension in a diploid that was also homozygous for citAΔ (Fig. 4D). Presumably, peroxisomal CitA resulting from citA3-34Δ can compensate for the loss of peroxisomal targeting due to the citAAK* mutation.
Deletion of citA results in greatly impaired growth on glucose-containing media but not on a variety of derepressing carbon sources. Expression of the mcsA gene encoding methylcitrate synthase is elevated under carbon-derepressing conditions. The creA204 mutation, which is derepressed for glucose repression (40), suppresses the glucose phenotype and results in derepression of mcsA. Therefore, McsA activity can replace the requirement for CitA, and this is supported by our inability to obtain a citAΔ mcsAΔ double mutant.
Consistent with these results, purified McsA from A. nidulans has been shown to have activity with both propionyl-CoA (Km, 1.7 μM) and acetyl-CoA (Km, 2.5 μM). The presence of potential CreA-binding sites in the 5′ end of mcsA suggested catabolite repression of expression (8). Purified McsA from A. fumigatus has a similar substrate specificity (31), and enzyme activity with propionyl-CoA as substrate and mcsA transcription is greatly increased by growth on peptone, a limiting carbon source (22). Analysis of the genomes of other filamentous fungi showed the presence of predicted mitochondria-targeted methylcitrate synthases (31). In P. anserina, deletion or loss-of-function mutants of the citrate synthase gene cit1 can grow on standard medium that contains dextrin, presumably a limiting carbon source, and 25% of wild-type citrate synthase activity is present in the mutants. Furthermore, Western blot assays showed a second weaker cross-reacting band in extracts from all strains when a polyclonal antibody to S. cerevisiae Cit1 was used (38). This additional citrate synthase is likely to be methylcitrate synthase, and a predicted gene is found in the P. anserina genome (PODANSg3467; accession no. CAP65504.1).
There is a third gene encoding a putative citrate synthase in A. nidulans (AN1079). This is more closely related to bacterial proteins than to CitA or McsA and lacks an obvious mitochondrial targeting sequence. There are related genes in other Aspergillus spp. with additional copies in the three sequenced A. niger strains (16, 44). These additional putative citrate synthases are not present in either N. crassa or Magnaporthe grisea (44). Our results indicate that this enzyme cannot substitute for CitA, and its function remains unknown.
Deletion of citA does not prevent growth on acetate, butyrate, or oleate, which are carbon sources generating acetyl-CoA and therefore requiring the glyoxylate cycle for growth. Citrate synthase activity is essential for this pathway, and therefore the mitochondrial McsA can substitute for CitA. Furthermore the citAAK* mutation, lacking the potential peroxisomal targeting sequence, does not prevent growth on these carbon sources. Therefore, peroxisomal activity is not required, despite the peroxisomal localization of the unique enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase in A. nidulans (21). This is consistent with the conclusion that glyoxylate cycle intermediates must be able to shuttle between cellular compartments in S. cerevisiae (26) and in C. albicans, which lacks peroxisomal citrate synthase. Unlike S. cerevisiae, in which peroxisomal Cit2 is redundant with the acetyl-carnitine shuttle (51, 52, 45), there is clearly no major role for CitA in the transfer of acetyl-CoA from peroxisomes into mitochondria. This is consistent with the absolute requirement for carnitine-acetyltransferases during growth on acetate or fatty acids for A. nidulans (42, 20).
Deletion of citA resulted in greatly reduced utilization of ethanol, which is metabolized via acetate to produce acetyl-CoA in the cytoplasm and therefore also requires the glyoxylate cycle. Microarray experiments show mcsA expression during growth on ethanol (10). A partial explanation for this result may be that the levels of mitochondrial acetyl-CoA resulting from ethanol catabolism are much lower than in acetate-grown cells and the lower citrate synthase activity of McsA is insufficient for adequate flux through the TCA cycle. McsA, as an essential enzyme of the methylcitrate cycle, is required for growth on propionate, and deletion of mcsA leads to sensitivity to the toxic effects of propionyl-CoA (5, 7, 8, 56). citAΔ resulted in increased growth on propionate and resistance to its toxicity. It is likely that, in propionate-grown wild-type strains, when both methylcitrate synthase and high citrate synthase activities are present, there is competition between the enzymes for the substrate oxaloacetate, leading to less conversion of propionyl-CoA to methylcitrate. Loss of CitA prevents this competition, allowing a balanced use of substrates and resulting in increased flux through both TCA and methylcitrate cycles. TCA cycle enzymes interact with each other and with the mitochondrial membrane (53, 41). This suggests that competition between CitA and McsA for binding to other TCA cycle enzymes (particularly aconitase) might reduce propionate utilization in the wild type. In the absence of CitA such competition would be eliminated.
Resting conidia of A. nidulans and A. fumigatus have been shown to contain stored mRNAs which are translated upon germination, and this also triggers transcription of specific genes (3, 27, 33, 34). It has also been suggested that germination results in a shift from a maintenance fermentative metabolism to respiration (27). Inhibitors of the respiratory chain severely delay germination of A. fumigatus conidia, and active mitochondria are found very early in germination (47). In microarray analysis of A. nidulans conidial germination, citA mRNA has been detected in ungerminated and germinated spores (3 and 5 h) while mcsA expression was not detected (deposited data of reference 3). Hybridization to A. fumigatus macroarrays of PCR fragments from approximately one-third of the genes showed that citrate synthase mRNA was present in resting spores and also increased during the first 30 min following germination, while methylcitrate synthase RNA, although detected before germination, was not present in the mRNA population at 30 min (27). We have detected mitochondrial localized CitA-GFP in both ungerminated conidia as well as 5 h after germination and found that germination of citAΔ conidia was significantly delayed. Loss of the putative CitA PTS1 did not affect germination. Of particular interest was the finding that the creA204 mutation suppressed the citAΔ germination defect, suggesting that derepressed mcsA expression could substitute for the loss of CitA. This implies that CreA-mediated carbon repression of mcsA expression operates during the synthesis of stored conidial mRNAs during conidial formation and/or during the early stages of germination. Upon germination, stored trehalose is rapidly mobilized (11) and may result in carbon repression. Studies with GFP-labeled McsA would be of interest.
A. nidulans is homothallic, and sexual development is promoted by glucose and low levels of aerobic respiration (18). We have found that sexual development is not affected by citAΔ, with normal production of Hulle cells, cleistothecia, and the red pigment, cleistothecin. This indicates that either there is no requirement for citrate synthase activity or that McsA is expressed in developing tissue and can provide sufficient activity. However, the cleistothecia produced in citAΔ selfed crosses were small and devoid of ascospores, and this is an autonomous property of cleistothecia produced in citAΔ × citA+ crosses. This shows that loss of citrate synthase activity results in a block in meiosis. Ascospore production was restored in the creA204 citAΔ double mutant, consistent with derepression of mcsA expression relieving the meiotic block. However, selfed crosses of citAΔ strains on the carbon sources lactose or arabinose did not produce ascospores, showing that derepression of mcsA expression in developing cleistothecia was not affected by the exogenous carbon source. These observations provide strong evidence for an intrinsic CreA-mediated carbon repression operating in a specific cell type, the ascogenous hyphae, within the developing cleistothecia. It has been proposed that, during sexual development, monosaccharides, released from cell walls by lytic enzymes produced by the Hulle cells, are used as the carbon source by the developing cleistothecia, and specific expression of a hexose transporter gene in ascogenous hyphae has been observed (54).
It has been found that amino acid starvation and defects in cross-pathway signaling of amino acid starvation leads to a block in sexual development in A. nidulans and other fungi (19). Amino acid biosynthesis could be blocked in citAΔ as a result of loss of intermediates generated by the TCA cycle. This explanation for our results seems unlikely, since the amino acid starvation block is at an earlier stage, with only poorly pigmented microcleistothecia, approximately 20 μm in diameter, formed, in contrast to the fully mature cleistothecia devoid of ascospores formed in citAΔ selfed crosses.
A requirement for citrate synthase activity during meiosis is compatible with observations in other fungi. In both Schizosaccharomyces pombe and S. cerevisiae, respiration is required throughout meiosis, while in S. cerevisiae it has been shown that a nonfermentable carbon source is required for the early stages of meiosis (23). In N. crassa inactivation of complex I of the respiratory chain by mutation results in an early block in meiosis (12). In contrast to A. nidulans, the heterothallic fungus P. anserina, like other Sordariomycetes, forms open fruiting bodies (perithecia) and is amenable to detailed studies of meiosis. Respiration mutants are female sterile (13). The effects of mutations in the citrate synthase gene, cit1, on sexual development in the heterothallic fungus P. anserina have been subject to detailed study (38). Immunofluorescence studies demonstrated the presence of mitochondrial localized protein in asci and ascospores. In homozygous crosses between most complete loss-of-function mutants of cit1, including a deletion, there is a block at a particular stage of meiotic prophase I as well as slowed pachytene and occasional defects in crozier formation. A cit1 mutation, resulting in the deletion of a single amino acid, produced only a partial loss of enzyme activity and did not affect meiosis. Both a mutation producing a truncated protein and an induced point mutation leading to inactive full-length protein had severe meiotic defects, but the specific block in meiotic prophase was not complete. A single amino acid substitution in a conserved domain almost completely lacks ascus development and shows no specific meiotic arrest, and this has been attributed to the mutant protein severely interfering with mitochondrial metabolism. A role for the Cit1 protein as opposed to the activity has been proposed. As noted above it is likely that in P. anserina citrate synthase activity is also provided by a methylcitrate synthase, and it is not known whether this is expressed during meiosis and the observed results are due to variable and incomplete interference with this activity by the different Cit1 variants. Our results do not support a role for CitA protein, as opposed to citrate synthase activity, in meiosis in A. nidulans.
The role of peroxisomal CitA is not at all clear. Deletion of the putative C-terminal PTS1 sequence does not affect carbon source utilization or developmental phenotypes, including germination. Furthermore, there does not seem to be a conserved role for peroxisomal citrate synthase in fungi, since M. grisea and C. albicans lack obvious PTS1 sequences (Fig. 3). Of course it cannot be excluded that there is an additional cryptic targeting sequence. It has been reported that Cit2 in S. cerevisiae can be targeted to both mitochondria and peroxisomes by a cryptic N-terminal targeting sequence that is unmasked when the PTS1 sequence is deleted (28). The sequences involved are not conserved in CitA. We have not been able to detect CitA-GFP-labeled peroxisomes in hyphae, conidia, or ascospores. This may represent a very low level of peroxisomal localization. In proteins with dual mitochondrial and peroxisomal targeting, it is common to find two ATG translation initiation signals, with one downstream of the mitochondrial targeting sequence, and these often arise from the use of two transcription start points. Examples include S. cerevisiae Cat2 (14, 52) and A. nidulans IdpA (46). There are no downstream ATG codons in citA that maintain the open reading frame, and mapped expressed sequence tags do not indicate alternative transcription start points. Therefore, any peroxisomal localization must result from import of either full-length protein or protein with the mitochondrial signal sequence removed. This is highly likely to result in very inefficient import and might account for our difficulty in detecting peroxisomal CitA-GFP. However, Penicillium chrysogenum CitA (which contains the PTS1 AKL) has been detected in purified peroxisomal proteins by liquid chromatography-tandem mass spectrometry analysis (25).
We found that the citAAK* mutation results in a reduced rate of colony extension arising from single ascospores and conidia. This phenotype was complemented by the citA3-34Δ mutation, which results in a protein retaining the PTS1 but with loss of mitochondrial targeting. In the citA3-34Δ-gfp strain, we observed rare punctate GFP localization in 5-h germinated conidia but not in hyphae, and this correlated with detection of some protein in this strain at this stage. It can therefore be suggested that a low level of peroxisomal CitA in germinated hyphae enhances colony growth. It is not obvious what role this activity plays in metabolism. While complete loss of peroxisomes in a pexC(3) mutant does not affect colony extension, loss of PTS1 protein targeting in a pexE(5)Δ does. Mutants with loss of the glyoxylate cycle or gluconeogenesis do not show this phenotype. Therefore, utilization of gluconeogenic substrates provided by mobilization of carbon sources stored in spores is unlikely. At present we do not have a satisfactory explanation for these data.
Overall we have shown that the putative CitA PTS1 sequence, which is conserved in other Aspergillus spp., does not play a major role in carbon source utilization or in the developmental functions of CitA. It is clear that peroxisomal citrate synthase activity is dispensable for the glyoxylate cycle. Furthermore, CitA function can be replaced by the citrate synthase activity of the mitochondria-localized methylcitrate synthase McsA when carbon catabolite repression is relieved, either by growth on derepressing carbon sources or by the loss of CreA repressor function. Of particular interest is the finding that derepression of mcsA due to the creA204 mutation, but not by derepressing carbon sources, suppresses the CitA requirement for the production of ascospores within cleistothecia. CreA has previously been regarded as responding to the externally supplied carbon source to regulate the transcription of genes required for the utilization of alternative carbon sources. Our results show that CreA-mediated repression can be an intrinsic property of the metabolism of particular cell types.
This work was supported by the Australian Research Council.
Assistance by Khanh Nguyen and Quentin Lang and provision of the mcsAΔ strain and helpful comments by Matthias Brock are gratefully acknowledged.
Published ahead of print on 19 February 2010.