Functional analysis of three putative galactofuranosyltransferases with redundant functions in galactofuranosylation in
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SHORT COMMUNICATION
Functional analysis of three putative galactofuranosyltransferases with redundant functions in galactofuranosylation in Aspergillus niger Mark Arentshorst1 · Davina de Lange1 · Joohae Park1 · Ellen L. Lagendijk1,2 · Ebru Alazi1,3 · Cees A. M. J. J. van den Hondel1 · Arthur F. J. Ram1 Received: 18 March 2019 / Revised: 5 July 2019 / Accepted: 20 July 2019 © The Author(s) 2019
Abstract Galactofuranose (Galf)-containing glycostructures are important to secure the integrity of the fungal cell wall. Golgi-localized Galf-transferases (Gfs) have been identified in Aspergillus nidulans and Aspergillus fumigatus. BLASTp searches identified three putative Galf-transferases in Aspergillus niger. Phylogenetic analysis showed that they group in three distinct groups. Characterization of the three Galf-transferases in A. niger by constructing single, double, and triple mutants revealed that gfsA is most important for Galf biosynthesis. The growth phenotypes of the ΔgfsA mutant are less severe than that of the ΔgfsAC mutant, indicating that GfsA and GfsC have redundant functions. Deletion of gfsB did not result in any growth defect and combining ΔgfsB with other deletion mutants did not exacerbate the growth phenotype. RT-qPCR experiments showed that induction of the agsA gene was higher in the ΔgfsAC and ΔgfsABC compared to the single mutants, indicating a severe cell wall stress response after multiple gfs gene deletions. Keywords Cell wall integrity · Galactofuranose · Galactomannan · Calcofluor white hypersensitive · Glycosylation · Golgi apparatus Abbreviations Galf Galactofuranose CFW Calcofluor white MM Minimal medium CM Complete medium
Communicated by Erko Stackebrandt. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00203-019-01709-w) contains supplementary material, which is available to authorized users. * Arthur F. J. Ram [email protected] 1
Institute of Biology Leiden, Molecular Microbiology and Biotechnology, Leiden University, Sylviusweg 72, 2333 BE Leiden, The Netherlands
2
Present Address: Koppert Biological Systems, Veilingweg 14, 2651 BE Berkel en Rodenrijs, The Netherlands
3
Present Address: Dutch DNA Biotech, Hugo R Kruytgebouw 4‑Noord, Padualaan 8, 3584 CH Utrecht, The Netherlands
Introduction Galactofuranose (Galf) is an important constituent of the fungal cell wall (Tefsen et al. 2012; Oka and Goto 2016). Around 5% of the dry weight of the cell wall of A. fumigatus consists of Galf (Lamarre et al. 2009) and similar amounts of Galf are expected to be present in other Aspergilli. Galf is the five-membered ring form of galactose and is found in several cell surface fractions. It has been identified as a component of the cell wall galactomannan fraction in Aspergilli, as a part of N- and O-glycans of extracellular proteins, and within glycosphingolipids (Bardalaye and Nordin 1977; Baretto-Bergter et al. 1980; Wallis et al. 1999; Toledo et al. 2007). We previously reported on the identification of severa
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