Metabolic and developmental effects resulting from deletion of the citA gene encoding citrate synthase in Aspergillus nidulans

Abstract

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 mcsADelta citADelta 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.

Fig. 1.

Methylcitrate synthase compensates for the loss of citrate synthase under conditions of carbon derepression. (A) Deletion of citA results in reduced growth on glucose. Colonies of the strains shown were inoculated onto glucose minimal medium and grown for 4 days at 37°C. The complemented strain contained a single copy of citA⁺ inserted at the wA locus, and the empty vector strain contained the wA-targeting vector alone inserted at the wA locus. The enlargement shows that citAΔ does not prevent conidiation. (B) Growth of citAΔ is equivalent to wild type on derepressing carbon sources except for ethanol. Carbon sources were added to minimal medium with 10 mM ammonium chloride as the nitrogen source at the following concentrations: glucose and peptone (1%); acetate, proline, γ-aminobutyric acid (GABA), and glutamate (50 mM); ethanol and quinate (0.5%); butyrate and oleate (10 mM). −, no added carbon source; CM, complete medium containing 1% glucose. (C) Suppression of the citAΔ phenotype by creA204. Medium was as for panel B, with xylose, fructose, lactose, and arabinose added at 1% and glycerol at 0.5%. Note that the creA204 mutation results in a compact colony morphology. (D) citAΔ is resistant to the toxic effects of propionate. Medium was as for panels B and C, with propionate added at the indicated concentrations. Note that mcsAΔ results in extreme sensitivity to propionate. In all cases (B to D) growth was for 2 to 3 days at 37°C. (E) Derepression of mcsA RNA levels, based on RT-PCR analysis of total RNA from wild-type (WT) and creA204 strains. RNA was extracted from mycelia grown in glucose minimal medium with ammonium tartrate as the nitrogen source and transferred to the indicated carbon sources for 4 h. Primers and cycle numbers are described in Materials and Methods. The tubulin gene (benA) was used as a loading control.

Fig. 2.

Effect of deletion of the potential peroxisome-targeting sequence of CitA. (A) C-terminal sequences of citrate synthase from various ascomycetes: S. cerevisiae Cit1 (YNR001C), Cit2 (YCR005C), and Cit3 (YPR001W); N.c, Neurospora crassa (NCU01692); P.a, P.anserina (CAC12961); M.g, Magnaporthe grisea (MGG_07202); Y. l, Yarrowia lipolytica (YALI0E02684g); C. alb, C. albicans (orf19.4393). McsA is methylcitrate synthase from A. nidulans. The asterisks above CitA indicate the residues that were replaced by stop codons. (B) Lack of effect of the citA^AK* mutation on growth. Growth tests were conducted as described for Fig. 1, with propionate present at 10 mM. The citA^AK*/citAΔ strain contains the citA^AK* mutant gene targeted to the wA locus in a citAΔ background, while the citA⁺/citA⁺strain contains citA⁺ targeted to the wA locus in a citA⁺ background.

Fig. 3.

Effects of citA mutations on sexual development and conidial germination. (A) Sexual development in selfings of the indicated genotypes. White arrows indicate cleistothecia. (B) Isolated cleistothecia. Bar, 273 μm. (C) Size distribution of cleistothecia from selfings of citA⁺and citAΔ and crosses between citA⁺and citAΔ strains, indicating a bimodal distribution of sizes. (D) Squashed cleistothecia from selfings, showing that citAΔ results in loss of ascospore production without affecting cleistothecin production. This phenotype is suppressed by the creA204 mutation. (E) Germination of conidia on glucose and acetate minimal media. Conidial dilutions were germinated, and images were captured with an inverted microscope. Conidia visibly germinated were counted and are expressed as a percentage of the total. At least 50 spores were counted, and values are the averages of at least three replicates with standard error bars shown.

Fig. 4.

Colony development is slower in the citA^AK* mutant. Spores were diluted, plated on complete medium, incubated at 37°C, and photographed at the indicated times. (A) Conidia; (B) ascospores from selfed strains. (C) Colony diameters (in mm) of the indicated strains resulting from plating conidia on complete medium were measured at the indicated times during incubation at 37°C. The same 10 colonies were counted at each time point. Standard error bars are shown. (D) Complementation in a diploid of the colony development phenotype of citA^AK* by citA^3-34Δ, encoding CitA lacking the mitochondria-targeting sequence. Conidia were plated on complete medium. (E) Colony development is slower in the pexE(5)Δ mutant. Ascospores from selfed pex mutants and conidia from pex mutants were plated and photographed as for panels A and B.

Fig. 5.

Localization and expression of CitA-GFP. (A) CitA-GFP is localized in mitochondria in hyphae. Mycelium was grown in 1% glucose minimal liquid medium for 16 h and then transferred for a further 4 h to the same medium or to minimal mediaum containing 50 mM acetate or 0.5% Tween 80 as a source of oleate. Mitochondria were visualized with MitoTracker Red CMXRos (Invitrogen). CitA-GFP and CitA^AK*-GFP are present in resting conidia (B) and resting ascospores (C). CitA^3-34Δ-GFP is not detectable. Bar, 10 μM (A and B) or 5 μM (C). (D) CitA^3-34Δ-GFP is visible as punctate dots in germinated conidia. Conidia were grown on coverslips in 1% glucose minimal liquid medium at 37°C for 5 h. Window, 10 μm. (E) Western blot of CitA-GFP-tagged strains on glucose and acetate. CitA^3-34Δ-GFP is not detectable in hyphae but is detectable at a low level in germinated conidia. Strains were grown as described for microscopy. Tubulin was used as a loading control.