Figure 1.
Multiple sequence alignment of the zinc coordinating region from fungal β-class CAs.
(A) ClustalX alignment was created using the following sequences: Sma1 [S. macrospora, Accession No. FM878639], Sma2 [FM878640], Sma3 [FM878641], Ncr1 [Neurospora crassa, Q7S631], Ncr2 [Q7S4J8], Ncr3 [Q8X0H0], Afu1 [Aspergillus fumigatus, Q4WQ18], Afu2 [A4DA32], Afu3 [Q4WPJ0], Cne1 [Cryptococcus neoformans, Q3I4V7], Cne2 [Q30E79], Sce [Saccharomyces cerevisiae, P53615], Spo [Schizosaccharomyces pombe, O94255] and Ror [Rhizopus oryzae, RO3G_10751.1]. Conserved amino acids important for Zn2+-coordination are marked by an asterisk. Identical amino acids, which are conserved in all proteins, are shaded in black; residues conserved in at least 13 of 15 sequences are shaded in dark grey and residues conserved in at least ten sequences are shaded in light grey. Arrows indicate S. macrospora cas genes with introns are given as grey boxes. The dashed box marks the region encoding the part of the protein which was used for the alignment at the top. The coding region for the mitochondrial target sequence of CAS2 is indicated as black box. (B) Amino acid identity in % is given for all sequences in pair-wise comparisons. Percentages given are based on amino acid comparison of the conserved region shown in (A).
Figure 2.
Expression analyses of cas genes.
Real time PCR was performed with total RNA isolated from S. macrospora wild type grown at 27°C in liquid BMM for three, five and seven days under ambient air conditions or 5% CO2. Comparisons are given as logarithmic values of the ambient air/5% CO2 ratios and are mean expression ratios from two independent biological replicates, each done in triplicate. For normalization, transcript levels of the SSUrRNA were calculated as described in Material and Methods. Asterisk indicate significance according to REST [70].
Figure 3.
Fluorescence microscopic analyses of S. macrospora wild type strains expressing cas1-egfp or cas3-egfp.
The images illustrate the fluorescence of CAS1-EGFP and CAS3-EGFP caused by the transformation of plasmids pGFP-CAS1 or pGFP-CAS3, respectively. Transformants were analyzed after growth for two days on solid SWG medium supplemented with hygromycin. DIC: differential interference contrast. Scale bar indicates 20 µm.
Figure 4.
Localization of the carbonic anhydrase CAS2.
(A) Fluorescence microscopic analysis of a S. macrospora wild type strain carrying plasmid pGFP-CAS2. Expression of full-length cas2-egfp leads to EGFP import into mitochondria, visible as tubular structures. CAS2-EGFP co-localizes to mitochondria stained with Mito-Tracker (Invitrogen, Germany) (B) Co-transformation of pGFP-CAS2 and pDsRED-SKL results in clearly distinguishable signals from green fluorescent mitochondria and red peroxisomes. (C) Fluorescence microscopic analysis of a S. macrospora wild type strain carrying either pMito-DsRED (N-terminal mitochondrial signal sequence of cas2 fused to DsRed) or pΔMito-CAS-DsRED (cas2 gene without the signal sequence fused to DsRed). DIC: differential interference contrast. Scale bar indicates 20 µm.
Figure 5.
RT-PCR analyses of wild type, single and double knock-out strains to confirm gene deletions.
The coding sequence of the cas genes were amplified with primer combinations cynT1-GFP-f/cynT1-r, cynT2-GFP-f/cynT2-r and cynT3-GFP-f/cynT3-r, respectively. Equal concentrations of DNA were loaded in each lane. Sizes of amplicons from genomic DNA control (gDNA) and cDNA is indicated aside. Control: negative control, without DNA template.
Figure 6.
Vegetative growth rate of S. macrospora wild type and cas mutant strains in ambient air and at elevated CO2.
Strains were grown for 3 days on solid BMM either in ambient air (black bars) or at 5% CO2 (white bars). Growth rate of wild type in ambient air was defined as 100%. Growth rates shown are averages from nine measurements of three independent experiments. Error bars are given as indicated.
Figure 7.
Fruiting-body developmental of wild type, single and double knock-out strains.
(A) Strains were grown on BMM medium for 7 days under natural environmental conditions (low CO2) or at high CO2 concentrations (5%). No morphological changes were observed in single and double Δcas1/3 and Δcas2/3 knock-out strains, respectively. Mature perithecia of Δcas1/2 strain in ambient air were not detectable after 7 days. Elevated CO2 concentrations complemented this phenotype. Scale bar indicates 500 µm (B) CO2/HCO3− accumulates in the fungal cell. Growth defect of Δcas2 is severely increased when mycelium is grown from germinated single spores (Δcas2_ssi: single spore isolate). BMM plate supplied with 0.5% sodium acetate was either inoculated with Δcas2 mutant strain or with an isolated single ascospore of Δcas2. Plates were incubated with or without CO2 for three days and images were taken from young mycelia. After prolonged incubation, Δcas2_ssi develops less fruiting-bodies under ambient air than a consecutively transferred mutant strain after nine days of growth. The defect is complemented at 5% CO2. Scale bar indicates 1 mm (C) Double knock-out mutant strain Δcas1/2 produced only few perithecia after 21 days of growth. Fruiting-bodies exhibit a wild-type phenotype, but are accumulated at several places on the plate. No mature ascospores were discharged from perithecia developed from Δcas1/2 strain.
Figure 8.
Germination rate of S. macrospora wild type and cas mutant strains.
(A) Ascospores were isolated from strains grown in ambient air. Only strain Δcas1/2 grew at 5% CO2 (marked by an asterisk); because it develops mature perithecia with asci and ascospores only at elevated CO2 levels (Figure 7). 100 single spores from 10 different perithecia of wild type and mutant strains were isolated and plated on BMM medium with 0.5% sodium acetate. Ascospores were incubated in ambient air or at 5% CO2. Germinated spores were counted after 2–5 days of incubation. The percentage of germination efficiency was determined for each strain. (B) Mycelia from germinated spores of Δcas2, Δcas1/2 and Δcas2/3 are affected in growth velocity and hyphal density. Ten spores were plated from each strain and images were taken after two days of growth. (C) Germlings of Δcas2 mutants are highly vacuolated. Spores were plated on glass slides overlaid with a thin layer of BMM supplied with 0.5% sodium acetate and incubated for one day. Vacuoles in hyphae of Δcas2 are marked by white arrows Scale bar indicates 50 µm.
Figure 9.
Complementation of the mutant phenotype of Δcas1/2 by ectopically integrated cas1, cas2 and cas3, respectively.
(A) Morphological characterization of Δcas1/2 strain complemented with different cas genes. The images show vegetative hyphae in case of Δcas1/2 strain grown in ambient air and fruiting-body development when grown at 5% CO2 or complemented with cas1, cas2, or cas3. The gene cas2 starts either with an ATG or CTG start codon; all constructs are driven by the constitutive gpd promoter of A. nidulans. Scale bar indicates 1 mm (B) At ambient air condition, mature ascospores were produced and discharged, only when Δcas1/2 is grown at elevated CO2-levels or complemented either with cas1 or ATG-cas2. After a prolonged incubation time of 18 days, complementation with cas3 also results in discharge of few mature ascospores. Scale bar indicates 50 µm (C) Germination rate and restoration of vegetative growth defects is partially complemented by constitutively expressed cas2 starting with ATG (+), but not by cas1, cas2-CTG and cas3 (—). Asterisk indicates that spores were isolated from a strain grown at 5% CO2, because no ascospores were developed in ambient air.
Table 1.
Sordaria macropsora strains used in this study