CAS2 belong to ‘‘plant’’-type b-CAs. Similar to ‘‘cab’’-type b- CAs, in CAS3, the amino acid residues corresponding to P. sativum Gln-151 are substituted with Pro, Ser-161 with Met, Ser-163 with Ala, Phe-179 with His, Val-184 with Ala and Tyr-205 with Val. Therefore, CAS3 can be classified as a ‘‘cab’’-type b-CA [36]. A further key difference between CAS1, CAS2 and CAS3 is the N-terminal extension of CAS2, which encodes for a mitochondrial target sequence. This is not a special feature of CAS2 from S. macrospora. CAs located in the mitochondria have been identified in algae, plants and animals [6,8,41–43]. A careful analysis of the complete genome sequences of other filamentous ascomycetes revealed that many of them encode one isoform with a mitochondrial target sequence. These sequences differ in length and exhibit very low sequence identity to the target sequence of S. macrospora [44]. The CAS2 homologue of N. crassa NCU08133.3 has been annotated without a mitochondrial target sequence and has been predicted to start with a CTG codon, a codon also conserved in S. macrospora CAS2. TargetP reliability indicated a lower likelihood of transport of the predicted protein NCU08133.3 into mitochondria. Since expression of a S. macrospora CTG variant of cas2 is not able to fully complement the phenotype of a Dcas1/2 deletion strain, we speculate that the genomic annotation of the N. crassa homologue is not correct. Re-annotation of the N. crassa NCU08133.3 by extending the first exon would lead to a protein with a mitochondrial target sequence [44]. Transcriptional regulation of b-CA genes in response to CO2 differs Three b-CA genes are simultaneously expressed in S. macrospora. Cas1 and cas3 are significantly regulated by CO2 on day three and five of development, whereas cas2 seems to be constitutively expressed during development (Figure 2). After three days of growth mRNA levels of cas1 and cas3 are significantly lower under ambient air conditions than under elevated CO2. While cas1 becomes up- regulated on day five under ambient air conditions, cas3 is no longer regulated by CO2. This result might implicate a specific function for CAS1 under CO2-limited conditions with regard to the production of sexual structures. Examination of the transcriptional regulation of fungal b-CA genes revealed that Nce103 of S. cerevisiae is up-regulated at low CO2 levels, whereas expression of C. albicans Nce103 and C. neoformans CAN2, encoding the major CA of C. neoformans, are not regulated by the CO2 concentration [12,16,45,46]. Evidence for a possible dependency between cas1 and cas2 is their transcriptional regulation in different cas knockout backgrounds. Whereas cas1 is not significantly regulated in a Dcas2 background, cas2 is completely de-regulated in Dcas1 and Dcas1/3 deletion strains. Although the Dcas1 strains do not exhibit any morpholog- ical phenotype, it seems that cas2 must be antagonistic to cas1. Similar to cas1, cas3 is only down-regulated at day three of sexual development but constitutively expressed under both conditions at later stages of development. However, in contrast to Dcas1 and Dcas2, growth of a Dcas3 strain is not influenced by elevated CO2 conditions (Figure 6). Interestingly, the transcrip- tional regulation is also not influenced by the presence or absence of the other two CA genes, indicating that cas3 might be involved in another bicarbonate dependent pathway that is independent from CO2 concentrations under the conditions tested. CAS2 is targeted to mitochondria and is required for germination and vegetative growth In many eukaryotes encoding multiple CAs, different isoforms of the enzyme have been shown to be distributed in different tissues and cell compartments [3]. When fused to egfp, the product b-Carbonic Anhydrases in Fungi of cas2 was exclusively targeted to the mitochondria, thus confirming the in silico prediction of a classical mitochondrial import sequence by TargetP and MitoProt II. While a-, b- and c- CAs have been reported to become targeted to mitochondria in mammals, plants and algae [3,6,8,42], this is the first time that a fungal b-CA has been shown to be located exclusively to mitochondria. No rigorous phenotypic changes related to sexual development were observed in CA single knockout strains. Only the Dcas2 mutant displayed a weak delay in vegetative growth, and this defect was complemented by the elevation of the CO2 concentration. Deletion of the cas2 gene may lead to a decrease in the mitochondrial bicarbonate concentration under ambient air conditions. In algae mitochondrial CAs are involved in the inorganic carbon- concentrating mechanism, supporting anaplerotic b-carboxylation and they are proposed to act as pH stabilizers [42,47,48]. Raven [49] hypothesized that if there is a HCO32 channel in the inner mitochondrial membrane, almost all of the inorganic carbon would leave the mitochondria as HCO32, thus limiting the potential for CO2 leakage through the plasmalemma. Similarly, mitochondrial b-CA CAS2 of S. macrospora may contribute to the cytoplasmic bicarbonate pool required for carboxylating enzymes relevant to many metabolic processes. Among these, acetyl-CoA carboxylase uses bicarbonate to produce malonyl-CoA for fatty acid synthesis. Indeed, it was shown recently that biosynthesis of fatty acids is affected by deletion of fungal CA genes. In the basidiomycete C. neoformans, the phenotype of a Dcan2 knockout was partially complemented by the addition of the fatty acid palmitate [16]. The growth defect of a S. cerevisiae Dnce103 strain under atmospheric pressure can be complemented only when end- products of bicarbonate-dependent metabolic pathways are sup- plied to the growth medium [31]. Complementation assays with a mixture of fatty acids also restored the vegetative growth defect of CA-deletion mutants in S. macrospora. In addition to the vegetative growth defect, the Dcas2 strain also displays a considerable decrease in germination efficiency, which was also obvious in Dcas2/3 and Dcas1/2 double deletion strains. S. macrospora appears to need a certain HCO32 concentration in the mitochondria for efficient ascospore germination. In contrast to vegetative growth defects, this defect of the Dcas2 mutant is not complemented by an elevated CO2 concentration. Therefore, we conclude that CAS2-mediated CO2/HCO32 homeostasis in mitochondria plays an important role in spore germination and in vegetative growth. Interestingly, the vegetative growth defect increased when colonies were derived from germinated spores. The mycelium derived from a germinating Dcas2 ascospore was composed of thin vacuolated hyphae indicating that CAS2 is needed at conditions of higher HCO32 demand (Figure 7B). Ascospores are dormant, metabolically-inactive cells, thus CO2 production through respiration is rather low and the HCO32 level is consequently low as well. We hypothesis that HCO32- dependent carboxylation reactions are needed for sufficient nutrient supply and energy recovery of the germinating ascospore. However, after a prolonged time of vegetative growth, HCO32 generated by CAS1 and/or CAS3 accumulates in the cell and partially abolishes the growth impairment. Taking our results into account, CAS3 appears not to be responsible for the HCO32 accumulation effect in the Dcas2 mutant strain. Dcas3 exhibited no obvious morphological phenotype under any conditions tested. Furthermore, a Dcas2/3 double knock-out strain behaved similarly to the Dcas2 mutant. HCO32 produced by CAS3 might be the co-factor for a reaction that is not linked to sexual reproduction, vegetative growth or PLoS ONE | www.plosone.org 11 April 2009 | Volume 4 | Issue 4 | e5177