b-Carbonic Anhydrases Play a Role in Fruiting Body Development and Ascospore Germination in the Filamentous Fungus Sordaria macrospora Skander Elleuche1,2, Stefanie Po¨ ggeler1* 1 Institute of Microbiology and Genetics, Department of Genetics of Eukaryotic Microorganisms, Georg-August University, Go¨ ttingen, Germany, 2 Faculty of Biology and Biotechnology, Ruhr-University of Bochum, Bochum, Germany Abstract Carbon dioxide (CO2) is among the most important gases for all organisms. Its reversible interconversion to bicarbonate (HCO32) 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 a-, b-, c-, d- and f-classes. While physiological functions of mammalian, prokaryotic, plant and algal CAs have been extensively studied over the past years, the role of b-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 b-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 (Dcas1, Dcas2, and Dcas3) 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 Dcas2 was affected in vegetative growth, fruiting-body development and ascospore germination, and the double mutant strain Dcas1/2 was completely sterile. Defects caused by the lack of cas2 could be partially complemented by elevated CO2 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. Citation: Elleuche S, Po¨ ggeler S (2009) b-Carbonic Anhydrases Play a Role in Fruiting Body Development and Ascospore Germination in the Filamentous Fungus Sordaria macrospora. PLoS ONE 4(4): e5177. doi:10.1371/journal.pone.0005177 Editor: Alfredo Herrera-Estrella, Cinvestav, Mexico Received January 8, 2009; Accepted March 12, 2009; Published April 13, 2009 Copyright: 2009 Elleuche, Po¨ ggeler. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The work was funded by the Deutsche Forschungsgemeinschaft grant number PO5332/3-2. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: spoegge@gwdg.de Introduction Carbon dioxide (CO2) and its hydration product bicarbonate (HCO32) are essential molecules in various physiological processes. In all domains of life, from microorganisms to mammals, CO2 is the end-product of respiration. Plants, algae and cyanobacteria are able to fix CO2 during photosynthesis. Although it represents only 0.036% of the atmospheric gases, CO2 is found at concentrations of roughly 5% in the human bloodstream and in tissues, where respiration takes place. CO2 is able to diffuse through lipid-containing cell membranes, but bicarbonate is negatively charged and not permeable to lipid bi- layers [1,2]. Since the reversible interconversion of CO2 and HCO32 is spontaneously balanced, CO2 is in equilibrium with HCO32 and carbonic acid. The reaction CO2+H2O « HCO32+H+ is slow, but can be accelerated by the ubiquitous enzyme carbonic anhydrase (CA; [EC 4.2.1.1]). CAs are typically Zn2+-metalloenzymes. Based on their amino acid sequence and structure they can be divided into five distinct classes, a, b, c, d and f, that share no sequence similarity, and appear to have evolved independently [3–5]. At least 16 different a- CA isoforms have been isolated in mammals, while plants and fungi encode a- and b-class carbonic anhydrases [3,6,7]. The c-class is predominantly distributed in archaea and eubacteria, but has also been identified in the mitochondria of plants [8,9]. The f- class, which has a cadmium center and the d-class have been discovered in marine diatoms [10,11]. To date, only ß-CAs have been characterized in fungi. The function of a b-CA in the hemiascomycetous yeasts Saccharomyces cerevisiae and in the human pathogen Candida albicans has been elucidated. In both, deletion of the b-CA-encoding NCE103 gene resulted in a mutant that required elevated CO2 levels for growth [12–14]. The S. cerevisiae mutant could be functionally comple- mented with a plant b-CA from Medicago sativa [14]. In addition, expression of the S. cerevisiae NCE103 gene was dramatically up- regulated under ambient air conditions [12]. In C. albicans the b- CA functions as a CO2 scavenger essential for pathogenicity in niches where the available CO2 is limited [13]. In contrast to S. cerevisiae and C. albicans, the basidiomycetous human pathogen Cryptococcus neoformans encodes two b-CAs whose functions have recently been elucidated [15,16]. Similar to C. albicans, growth and morphogenesis of C. neoformans is strongly influenced by CO2, which is directly sensed by the bicarbonate-regulated adenylyl cyclases [13,15]. The CAN2 gene of C. neoformans was shown to PLoS ONE | www.plosone.org 1 April 2009 | Volume 4 | Issue 4 | e5177