Transcription levels of CHS5 and CHS4 genes in Paracoccidioides brasiliensis mycelial phase, respond to alterations in external osmolarity, oxidative stress and glucose concentration Gustavo A. NIN ˜ O-VEGA*, Franc ¸oise SORAIS, Gioconda SAN-BLAS Instituto Venezolano de Investigaciones Cientı´ficas (IVIC), Centro de Microbiologı´a y Biologı´a Celular, Apartado 20632, Caracas 1020A, Venezuela article info Article history: Received 24 March 2009 Received in revised form 7 July 2009 Accepted 9 July 2009 Available online 17 July 2009 Corresponding Editor: Daniel C. Eastwood Keywords: CHS5 Glucose repression Hypo-osmotic stress Myosin motor domain abstract The complete sequence of Paracoccidioides brasiliensis CHS5 gene, encoding a putative chitin synthase revealed a 5583 nt open reading frame, interrupted by three introns of 82, 87 and 97 bp (GenBank Accession No EF654132). The deduced protein contains 1861 amino acids with a predicted molecular weight of 206.9 kDa. Both its large size and the presence of a N-terminal region of approx. 800 residues with a characteristic putative myosin motor- like domain, allow us to include PbrChs5 into class V fungal chitin synthases. Sequence analysis of over 4 kb from the 5 0 UTR region in CHS5, revealed the presence of a previously reported CHS4 gene in P. brasiliensis, arranged in a head-to-head configuration with CHS5. A motif search in this shared region showed the presence of stress response elements (STREs), three binding sites for the transcription activators Rlm1p (known to be stimulated by hypo-osmotic stress) and clusters of Adr1 (related to glucose repression). A quantitative RT-PCR analysis pointed to changes in transcription levels for both genes following oxida- tive stress, alteration of external osmolarity and under glucose-repressible conditions, sug- gesting a common regulatory mechanism of transcription. ª 2009 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. Introduction The fungal wall is a structure that provides protection to the cell, acting as an initial barrier against hostile environments while, at the same time, holding up the cell integrity against its internal turgor pressure. It is a dynamic structure, which could change in composition and structural organization as the cell grows and/or modifies its morphology. These changes are tightly regulated during the cell cycle and in response to changing environmental conditions, stress and mutations in cell wall biosynthetic processes (Klis et al. 2006; Ruiz-Herrera et al. 2006). In yeasts, any stress on the cell wall leads to compensatory responses (Popolo et al. 2001) such as the up- regulation of chitin synthesis, among others. Chitin is one major structural component of the fungal cell wall. It has important functions in wall integrity (Fujiwara et al. 1997; Wang & Szaniszlo 2000), morphogenesis (Cabib et al. 1988) and conidiophore development (Aufauvre-Brown et al. 1997; Fujiwara et al. 2000). Exposure of Candida albicans to cell wall stress by Calcofluor White or CaCl 2 induces an increase in both the in vitro chitin synthase activity and the amount of chitin in the cell wall (Munro et al. 2007). Chitin synthesis in fungi is regulated by multigene families encoding chitin synthase isoenzymes, some of them * Corresponding author. Tel.: 58 212 504 1364; fax: 58 212 504 1382. E-mail address: gnino@ivic.ve journal homepage: www.elsevier.com/locate/mycres mycological research 113 (2009) 1091–1096 0953-7562/$ – see front matter ª 2009 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2009.07.005