b-Carbonic Anhydrases in Fungi Figure 9. Complementation of the mutant phenotype of Dcas1/2 by ectopically integrated cas1, cas2 and cas3, respectively. (A) Morphological characterization of Dcas1/2 strain complemented with different cas genes. The images show vegetative hyphae in case of Dcas1/2 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 Dcas1/2 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 mm (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. doi:10.1371/journal.pone.0005177.g009 fungi. Despite being well-characterized in prokaryotes, plants and animals, CAs have been analyzed in only three fungal species [12,13,16,32,33]. The data presented here demonstrate that three b-CAs are encoded within the genome of the filamentous ascomycete S. macrospora. To the best of our knowledge, this is the first genetic analysis on the function of b-CAs in filamentous ascomycetes. S. macrospora has three b-CAs The proteins encoded by cas1, cas2 and cas3 showed a significant degree of sequence identity when compared to known b-CAs of bacterial and plant origin. The crystal structures of b-CAs from plants, bacteria, the archaeon Methanobacterium thermoautotrophicum and C. neoformans revealed that the active site zinc is coordinated by two conserved cysteines and one conserved histidine [32,34–38]. In addition to these three residues, an aspartic acid and an arginine are also structurally conserved in every b-CA sequence known [4]. These two residues presumably assist in a number of catalytic steps, including substrate binding, proton shuffling and product release, or act as the fourth zinc ligand [32,35,39]. Sequence alignment demonstrated that these five residues are also conserved in CAS1, CAS2 and CAS3 (Figure 1A). Based on other key active site residues, b-CAs can be further divided into ‘‘plant’’- type b-CAs, including all known sequences of plants and of some bacteria and into ‘‘cab’’-type enzymes of M. thermoautotrophicum, Mycobacterium tuberculosis and Bacillus subtilis and other bacteria and archaea [36,40]. Active site residues corresponding to key active site residues Gln-151, Ser-161, Ser-163, Phe-179, and Tyr-205 of the Pisum sativum b-CA [36] are conserved in CAS1 and CAS2. Only Val-184 is replaced by Ile in CAS1 as well as in CAS2. However, a survey of other plant b-CA sequences indicated that either Ile or Val is found at this position [36]. Thus, CAS1 and PLoS ONE | www.plosone.org 10 April 2009 | Volume 4 | Issue 4 | e5177