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Beta-carbonic anhydrases play a role in fruiting body development and ascospore germination in the filamentous fungus Sordaria macrospora.

Elleuche S, Pöggeler S - PLoS ONE (2009)

Bottom Line: No effect on vegetative growth, fruiting-body and ascospore development was seen in the single mutant strains lacking cas1 or cas3, while single mutant Deltacas2 was affected in vegetative growth, fruiting-body development and ascospore germination, and the double mutant strain Deltacas1/2 was completely sterile.Defects caused by the lack of cas2 could be partially complemented by elevated CO(2) levels or overexpression of cas1, cas3, or a non-mitochondrial cas2 variant.The results suggest that CAs are required for sexual reproduction in filamentous ascomycetes and that the multiplicity of isoforms results in redundancy of specific and non-specific functions.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology and Genetics, Department of Genetics of Eukaryotic Microorganisms, Georg-August University, Göttingen, Germany.

ABSTRACT
Carbon dioxide (CO(2)) is among the most important gases for all organisms. Its reversible interconversion to bicarbonate (HCO(3) (-)) reaches equilibrium spontaneously, but slowly, and can be accelerated by a ubiquitous group of enzymes called carbonic anhydrases (CAs). These enzymes are grouped by their distinct structural features into alpha-, beta-, gamma-, delta- and zeta-classes. While physiological functions of mammalian, prokaryotic, plant and algal CAs have been extensively studied over the past years, the role of beta-CAs in yeasts and the human pathogen Cryptococcus neoformans has been elucidated only recently, and the function of CAs in multicellular filamentous ascomycetes is mostly unknown. To assess the role of CAs in the development of filamentous ascomycetes, the function of three genes, cas1, cas2 and cas3 (carbonic anhydrase of Sordaria) encoding beta-class carbonic anhydrases was characterized in the filamentous ascomycetous fungus Sordaria macrospora. Fluorescence microscopy was used to determine the localization of GFP- and DsRED-tagged CAs. While CAS1 and CAS3 are cytoplasmic enzymes, CAS2 is localized to the mitochondria. To assess the function of the three isoenzymes, we generated knock-out strains for all three cas genes (Deltacas1, Deltacas2, and Deltacas3) as well as all combinations of double mutants. No effect on vegetative growth, fruiting-body and ascospore development was seen in the single mutant strains lacking cas1 or cas3, while single mutant Deltacas2 was affected in vegetative growth, fruiting-body development and ascospore germination, and the double mutant strain Deltacas1/2 was completely sterile. Defects caused by the lack of cas2 could be partially complemented by elevated CO(2) levels or overexpression of cas1, cas3, or a non-mitochondrial cas2 variant. The results suggest that CAs are required for sexual reproduction in filamentous ascomycetes and that the multiplicity of isoforms results in redundancy of specific and non-specific functions.

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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).
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pone-0005177-g001: 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).

Mentions: S. macrospora and N. crassa are closely related and share a high degree of nucleic acid identity within open reading frames. Therefore, the N. crassa database is often used to identify homologous genes in S. macrospora, whose genome is not yet sequenced [24]. Database searches with the S. cerevisiae CA NCE103p revealed the presence of two putative CA genes in the genome of N. crassa. The enzymes are encoded by the ORFs NCU04778.3 and NCU08133.3. A BLASTP search using the catalytic region of the putative CA NCU08133.3 resulted in the identification of a third CA encoded by NCU01103.3. Using heterologous primers designed from the N. crassa CA ORFs, we succeeded in isolating three putative CA genes from S. macrospora. Sequence alignments with several fungal CAs assigned the three S. macrospora CAs to the β-class. Three highly conserved residues function as zinc-binding ligands (Figure 1A). Based on these homologies, the three S. macrospora genes were termed cas1 (carbonic anhydrase of Sordaria macrospora; NCU04778.3 homologue, accession number FM878639), cas2 (NCU08133.3 homologue, accession number FM878640) and cas3 (NCU01103.3 homologue, accession number FM878641). The conserved β-CA domain is located in the central part of CAS1 and CAS3, and in the C terminus of CAS2 (Figure 1A). In cas3, the coding region of the conserved domain is interrupted by an intron. To confirm the splicing of predicted introns, cDNAs of the three cas genes were generated by RT-PCR with primer pairs cynT1-pQE-f/cynT1-r; cynT2-pQE-f/cynT2-r and cynT3-pQE-f/cynT3-r, respectively. We confirmed the presence of one predicted intron within the 770 bp coding region of the cas1 gene (65 bp), and two predicted introns within the 733 bp coding region of the cas3 gene (103 and 105 bp). Sequence analysis of the 855 bp cas2 coding region revealed that this gene is not interrupted by an intron. The deduced peptide sequences of the putative S. macrospora cas1, cas2 and cas3 cDNAs encode for 234, 284 and 174 aa proteins with predicted molecular masses of 25.1, 32.1 and 19.1 kDa, respectively, and theoretical isoelectric points of 6.16, 8.15 and 5.16. cDNAs of the three S. macrospora cas genes fused to either RGS-His-tag or GST-tag were heterologously expressed in E. coli. SDS-PAGE and Western blot analyses with anti-RGS-His and anti-GST antibodies revealed protein bands with apparent molecular weights consistent with the calculated molecular weights of the S. macrospora CAs (data not shown).


Beta-carbonic anhydrases play a role in fruiting body development and ascospore germination in the filamentous fungus Sordaria macrospora.

Elleuche S, Pöggeler S - PLoS ONE (2009)

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).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2664464&req=5

pone-0005177-g001: 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).
Mentions: S. macrospora and N. crassa are closely related and share a high degree of nucleic acid identity within open reading frames. Therefore, the N. crassa database is often used to identify homologous genes in S. macrospora, whose genome is not yet sequenced [24]. Database searches with the S. cerevisiae CA NCE103p revealed the presence of two putative CA genes in the genome of N. crassa. The enzymes are encoded by the ORFs NCU04778.3 and NCU08133.3. A BLASTP search using the catalytic region of the putative CA NCU08133.3 resulted in the identification of a third CA encoded by NCU01103.3. Using heterologous primers designed from the N. crassa CA ORFs, we succeeded in isolating three putative CA genes from S. macrospora. Sequence alignments with several fungal CAs assigned the three S. macrospora CAs to the β-class. Three highly conserved residues function as zinc-binding ligands (Figure 1A). Based on these homologies, the three S. macrospora genes were termed cas1 (carbonic anhydrase of Sordaria macrospora; NCU04778.3 homologue, accession number FM878639), cas2 (NCU08133.3 homologue, accession number FM878640) and cas3 (NCU01103.3 homologue, accession number FM878641). The conserved β-CA domain is located in the central part of CAS1 and CAS3, and in the C terminus of CAS2 (Figure 1A). In cas3, the coding region of the conserved domain is interrupted by an intron. To confirm the splicing of predicted introns, cDNAs of the three cas genes were generated by RT-PCR with primer pairs cynT1-pQE-f/cynT1-r; cynT2-pQE-f/cynT2-r and cynT3-pQE-f/cynT3-r, respectively. We confirmed the presence of one predicted intron within the 770 bp coding region of the cas1 gene (65 bp), and two predicted introns within the 733 bp coding region of the cas3 gene (103 and 105 bp). Sequence analysis of the 855 bp cas2 coding region revealed that this gene is not interrupted by an intron. The deduced peptide sequences of the putative S. macrospora cas1, cas2 and cas3 cDNAs encode for 234, 284 and 174 aa proteins with predicted molecular masses of 25.1, 32.1 and 19.1 kDa, respectively, and theoretical isoelectric points of 6.16, 8.15 and 5.16. cDNAs of the three S. macrospora cas genes fused to either RGS-His-tag or GST-tag were heterologously expressed in E. coli. SDS-PAGE and Western blot analyses with anti-RGS-His and anti-GST antibodies revealed protein bands with apparent molecular weights consistent with the calculated molecular weights of the S. macrospora CAs (data not shown).

Bottom Line: No effect on vegetative growth, fruiting-body and ascospore development was seen in the single mutant strains lacking cas1 or cas3, while single mutant Deltacas2 was affected in vegetative growth, fruiting-body development and ascospore germination, and the double mutant strain Deltacas1/2 was completely sterile.Defects caused by the lack of cas2 could be partially complemented by elevated CO(2) levels or overexpression of cas1, cas3, or a non-mitochondrial cas2 variant.The results suggest that CAs are required for sexual reproduction in filamentous ascomycetes and that the multiplicity of isoforms results in redundancy of specific and non-specific functions.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology and Genetics, Department of Genetics of Eukaryotic Microorganisms, Georg-August University, Göttingen, Germany.

ABSTRACT
Carbon dioxide (CO(2)) is among the most important gases for all organisms. Its reversible interconversion to bicarbonate (HCO(3) (-)) reaches equilibrium spontaneously, but slowly, and can be accelerated by a ubiquitous group of enzymes called carbonic anhydrases (CAs). These enzymes are grouped by their distinct structural features into alpha-, beta-, gamma-, delta- and zeta-classes. While physiological functions of mammalian, prokaryotic, plant and algal CAs have been extensively studied over the past years, the role of beta-CAs in yeasts and the human pathogen Cryptococcus neoformans has been elucidated only recently, and the function of CAs in multicellular filamentous ascomycetes is mostly unknown. To assess the role of CAs in the development of filamentous ascomycetes, the function of three genes, cas1, cas2 and cas3 (carbonic anhydrase of Sordaria) encoding beta-class carbonic anhydrases was characterized in the filamentous ascomycetous fungus Sordaria macrospora. Fluorescence microscopy was used to determine the localization of GFP- and DsRED-tagged CAs. While CAS1 and CAS3 are cytoplasmic enzymes, CAS2 is localized to the mitochondria. To assess the function of the three isoenzymes, we generated knock-out strains for all three cas genes (Deltacas1, Deltacas2, and Deltacas3) as well as all combinations of double mutants. No effect on vegetative growth, fruiting-body and ascospore development was seen in the single mutant strains lacking cas1 or cas3, while single mutant Deltacas2 was affected in vegetative growth, fruiting-body development and ascospore germination, and the double mutant strain Deltacas1/2 was completely sterile. Defects caused by the lack of cas2 could be partially complemented by elevated CO(2) levels or overexpression of cas1, cas3, or a non-mitochondrial cas2 variant. The results suggest that CAs are required for sexual reproduction in filamentous ascomycetes and that the multiplicity of isoforms results in redundancy of specific and non-specific functions.

Show MeSH
Related in: MedlinePlus