ascospore germination. In this context, it would be interesting to investigate a possible connection between CA mediated HCO32 production and cyanate decomposition, a reaction that in S. macrospora, has been recently shown to be catalyzed by a bicarbonate-dependent cyanase. Deletion of the cyanase-encoding gene cyn1 does not lead to any defects in vegetative growth or fruiting-body development [50]. In E. coli the b-class CA CynT is encoded in an operon together with the cyanase-encoding gene cynS, and the function of the E. coli CA appears to be to prevent HCO32 depletion during cyanate decomposition [51]. Deletion of cas1 and cas2 blocks sexual reproduction The Dcas1/2 double deletion strain displays a more severe phenotype than the Dcas2 single knockout strain and the Dcas2/3 double knockout strain, indicating that in addition to CAS2, CAS1 is also an active CA. While perithecia produced by S. macrospora wild type contain about 100 asci in ambient air [52], the production of fruiting-bodies and differentiated ascus rosettes is drastically impaired in the Dcas1/2 double deletion strain. A small number of fruiting-bodies with only immature asci was produced after 21 days of growth. The defect could be partially comple- mented by incubation at elevated CO2 levels, which resulted in the production of fruiting-bodies containing significantly fewer mature ascospores than the wild type (Figure 7 and Figure 9). The phenotype of the Dcas1/2 mutant resembles the S. macrospora developmental mutant per5, which produces only ascus precursors without any mature spores. The per5 mutant has been shown to encode a defective ATP-citrate lyase that is involved in the formation of acetyl-CoA used for the biosynthesis of fatty acids [53]. In contrast to the complementation of the vegetative growth defect, the impaired production of mature asci in the Dcas1/2-fus was not reversed by fatty acids. This result indicates that specific CA activity impacts further biochemical pathways than fatty acid biosynthesis during sexual development. Thus, in 5% CO2, the small amount of bicarbonate generated through nonenzymatic, spontaneous hydration of CO2 and/or through the activity of the remaining CAS3 is sufficient to support fruiting-body and ascospore formation. Similarly a Dcan2 mutant of C. neoformans was shown to exhibit a severe sporulation defect [16]. However, it should be taken into account that complementation of Dcas1/2-fus strain by overexpression of cas1, cas2 and cas3 due to the use of a strong heterologous gpdA promoter of A. nidulans could increase some paths and decrease others, consequently affecting the overall phenotype. These results demonstrated that the protein encoded by cas3 has CA activity, albeit it is probably less active than CAS1 and CAS2. Growth defects caused by the deletion of CA genes in bacteria, have been shown to be rescued by introducing heterologous CA genes [54,55]. The growth defect of a S. cerevisiae CA-mutant strain was complemented by plant, plant chloroplast, human, prokary- otic and fungal CAs [14,32,56,57]. Apparently, in these unicellular organisms, only a small amount of CA activity is sufficient for growth at ambient air conditions, irrespective of its origin, whereas in S. macrospora, mitochondrial targeting of CAS2 seems to also be essential for growth and sexual reproduction. In summary, complementation assays in combination with phenotypic observations indicate that in S. macrospora cas2 and possibly also cas1 may encode for a major CA involved in fruiting- body and ascospore development, as well as in ascospore germination, while cas3 encodes for a minor CA. This must be confirmed by in vitro activity measurements, however, as recently shown for the C. neoformans and the C. albicans CAs [58]. Furthermore, analysis in C. albicans and C. neoformans revealed a functional link between cAMP signaling and CO2/HCO32 b-Carbonic Anhydrases in Fungi sensing and showed that bicarbonate directly activates adenylyl cyclase [7,13,15]. Similar to the phenotypes presented here, deletion of adenylyl cyclase in S. macrospora has recently been shown to affect vegetative growth and sexual development in S. macrospora [19]. In this respect, further examination of the functional connection between CAs and adenylyl cyclase in fruiting-body and ascospore formation of filamentous ascomycetes will be of great interest. Materials and Methods Strains, media and culture conditions Cloning and propagation of recombinant plasmids was done under standard conditions with Escherichia coli strain SURE used as a host for plasmid amplification [59]. Genotypes of S. macrospora strains used and generated in this study are provided in Table 1. All strains were cultivated either on solid or in liquid corn meal medium (BMM) or in liquid complete medium (CM) or on solid SWG medium [23], incubated in ambient air or at 5% carbon dioxide in the Inkubator C42 (Labotect, Germany). Transforma- tion of S. macrospora was performed as described previously with 20 mg/ml Glucanex 200G (Schliessmann, Germany) to degrade fungal cell walls [53]. The determination of germination rates were conducted by isolating 100 single ascospores. After inoculation for 2–5 days on BMM supplied with 0.5% of sodium acetetate, the number of germinated spores was determined. Dry cell weight was measured according to Nolting and Po¨ggeler [60] and growth velocities were either determined according to Nowrousian and Cebula [61], or by measuring the colony radius on agar plates after 1 to 10 days. For complementation assays with fatty acids, 30 mg/l of palmitic, myristic and stearic acid and 1% Tween 80 were added to BMM medium according to Aguilera et al. [31]. Genomic DNA was isolated from cultures grown for 3 days in liquid CM medium; RNA samples for qRT-PCR were isolated from cultures grown for 5 days in liquid CM or BMM. Sequence analysis Primers were synthesized at MWG Biotech AG (Ebersberg, Germany). DNA sequencing was performed by the sequencing service of the Department of Biochemistry (Ruhr-University of Bochum, Germany) or by the G2L-sequencing service of the Go¨ ttinger Genom Labor (Georg-August University of Go¨ ttingen, Germany) or by MWG Biotech Customer Service (Ebersberg, Germany). Molecular weights and isoelectric points of proteins were calculated with programs from the ExPASy Proteomics Server (http://www.expasy.org). Protein sequence alignments were performed using the ClustalX program [62]. Protein and nucleotide sequence data of b-CAs from other organisms were obtained from the public databases at NCBI (http://www.ncbi. nlm.nih.gov/entrez/) or by BLAST searches of the complete sequenced genomes at the Broad Institute (http://www.broad.mit. edu/annotation/fungi/fgi/). Subcellular localization of carbonic anhydrases was predicted with the TargetP program provided on the CBS Prediction Server (http://www.cbs.dtu.dk/services/) and with MitoProt II - v1.101 [29]. Preparation of nucleic acids Isolation of genomic DNA from S. macrospora was done as described previously [22]. RNA was isolated from mycelial samples, which were three times incubated in liquid nitrogen for 5 sec. Afterwards the frozen sample was supplemented with 1 ml Trizol (Invitrogen) and homogenized two times (2 min, 1000 Hz) using the TissueLyser (Qiagen), followed by centrifugation PLoS ONE | www.plosone.org 12 April 2009 | Volume 4 | Issue 4 | e5177