encode its major CA. Its activity is essential to survival and proliferation as well as for basidia and basidiospore formation, but is dispensable for lethality during infection [16]. Similar to C. neoformans, filamentous ascomycetes seem to contain multiple b-CAs: three b-CA homologues have been identified in the genome of the ascomycete Aspergillus fumigatus [7]. However, the functional role of b-CAs in filamentous fungi has not yet been assessed and it is still not known if the multiplicity of isoforms is related to organelles or cell- or tissue- specific expression and function during development. Hence, we were interested in assessing the functional role of CAs in filamentous ascomycetes. Here, we present a genetic analysis of three b-CAs from the model organism Sordaria macrospora, which has been intensively used to study sexual reproduction and fruiting-body (perithecium) differentiation [17–22]. S. macrospora is a coprophilous fungus that lives on the dung of herbivores. It is a close relative of Neurospora crassa, but is in contrast to the heterothallic (self-sterile) N. crassa, homothallic (self-fertile) [23,24]. Under laboratory conditions, S. macrospora completes its life cycle within seven days and produces pear-shaped fruiting-bodies with asci and ascospores. A genetic advantage of S. macrospora is that recessive mutations can directly be tested for impairment in fruiting-body development, without the need of crossing strains of opposite mating-types [25]. In this study we identified three genes encoding b-CAs (cas1, cas2 and cas3) in S. macrospora. Localization studies revealed that CAS2 was translocated into the mitochondria, while CAS1 and CAS3 are cytoplasmic. A genetic analysis of knock-out strains, Dcas1, Dcas2, and Dcas3, demonstrate that CAS2 may be the major CA in S. macrospora, involved in vegetative growth and ascospore germination. Developmental defects were severely increased in a Dcas1/2 double deletion strain, which was completely sterile and produced a reduced number of empty fruiting-bodies only after 21 days of growth. The defect in fruiting-body development, but not the decrease in germination efficiency, could be complemented by elevated CO2 (5%) concentrations. Interestingly, we were also able to complement the delay in fruiting-body formation in the Dcas1/ 2 strain by overexpressing either cas1, cas3, or a cytoplasmically- located cas2 variant, indicating that the function of CAs is redundant for fruiting-body formation in S. macrospora. Results Identification of three carbonic anhydrase genes in S. macrospora 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 b-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 homo- logue, accession number FM878639), cas2 (NCU08133.3 homo- b-Carbonic Anhydrases in Fungi logue, accession number FM878640) and cas3 (NCU01103.3 homologue, accession number FM878641). The conserved b-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). When comparing the entire amino acid (aa) sequence of the b- CAs of S. macrospora and N. crassa, all enzymes shared a high degree of aa sequence similarities. BLASTP analysis of the identified proteins also revealed high degrees of sequence similarities to a recently characterized fungal b-CA from the pathogenic yeast C. albicans, two CAs of the pathogenic basidiomycete Cryptococcus neoformans as well as to non-characterized homologues from other ascomycetes and the zygomycete Rhizopus oryzae (Figure 1A+B). Expression of cas genes in S. macrospora The CA Nce103p of S. cerevisiae is induced by low CO2 levels, whereas C. albicans Nce103p is constitutively expressed [12,13]. To understand the functions of CAs in the filamentous fungus S. macrospora, we investigated the transcriptional expression of cas1, cas2 and cas3 in the wild type grown in ambient air or with exposure to 5% CO2, by real time PCR. In all experiments, cas1 mRNA levels were significantly lower under ambient air conditions after three days of development and up-regulated after five days whereas cas2 transcript was not significantly regulated under the conditions investigated. Expression of cas3 was significantly down-regulated under ambient air conditions after three days of growth and not regulated by different CO2 conditions after five and seven days (Figure 2). Subcellular distribution of carbonic anhydrases To gain a better understanding of the cellular localization of CAs in S. macrospora, we performed fluorescence microscopy experiments and transformed plasmids pCAS1-GFP, pCAS2-GFP and pCAS3-GFP carrying cas-egfp fusion genes under control of the strong constitutive gpd promoter of A. nidulans, into the S. macrospora wild type. According to an in silico TargetP and MitoProt II - v1.101 analysis, CAS2 was predicted to be located in the mitochondria, whereas CAS1 and CAS3 were predicted to be cytoplasmic. Microscopic analysis of the transformants revealed that CAS1-EGFP and CAS3-EGFP proteins were uniformly distributed throughout the cytoplasm of fast-growing, young hyphae, as well as old hyphae. Fluorescence was not concentrated in any organelle and was absent from vacuoles (Figure 3). The CAS2-EGFP fusion protein clearly localized to tubular structures throughout young hyphae and hyphal tips (Figure 4A). Tubular or round-shaped structures are typical for mitochondria in fungal PLoS ONE | www.plosone.org 2 April 2009 | Volume 4 | Issue 4 | e5177