Abstract
Radial growth, asexual sporulation, and cleistothecia formation as well as extracellular chitinase and proteinase formation of Aspergillus nidulans were monitored in surface cultures in order to study the physiological role of extracellular hydrolase production in carbon-stressed cultures. We set up carbon-stressed and carbon-overfed experimental conditions by varying the starting glucose concentration within the range of 2.5 and 40 g/L. Glucose starvation induced radial growth and hydrolase production and enhanced the maturation of cleistothecia; meanwhile, glucose-rich conditions enhanced mycelial biomass, conidia, and cleistothecia production. Double deletion of chiB and engA (encoding an extracellular endochitinase and a β-1,3-endoglucanase, respectively) decreased conidia production under carbon-stressed conditions, suggesting that these autolytic hydrolases can support conidia formation by releasing nutrients from the cell wall polysaccharides of dead hyphae. Double deletion of prtA and pepJ (both genes encode extracellular proteases) reduced the number of cleistothecia even under carbon-rich conditions except in the presence of casamino acids, which supports the view that sexual development and amino acid metabolism are tightly connected to each other in this fungus.
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References
Adams TH, Wieser JK, Yu JH (1998) Asexual sporulation in Aspergillus nidulans. Microbiol Mol Biol Rev 62:35–54
Amoah-Buahin E, Bone N, Armstrong J (2005) Hyphal growth in the fission yeast Schizosaccharomyces pombe. Eukaryot Cell 4:1287–1297. https://doi.org/10.1128/EC.4.7.1287-1297.2005
Atoui A, Kastner C, Larey CM, Thokala R, Etxebeste O, Espeso EA, Fischer R, Calvo AM (2010) Cross-talk between light and glucose regulation controls toxin production and morphogenesis in Aspergillus nidulans. Fungal Genet Biol 47:962–972. https://doi.org/10.1016/j.fgb.2010.08.007
Barratt RW, Johnson GB, Ogata WN (1965) Wild-type and mutant stocks of Aspergillus nidulans. Genetics 52:233–246
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 2:248–254
Broach JR (2012) Nutritional control of growth and development in yeast. Genetics 192:73–105. https://doi.org/10.1534/genetics.111.135731
Brown NA, de Gouvea PF, Krohn NG, Savoldi M, Goldman GH (2013) Functional characterisation of the non-essential protein kinases and phosphatases regulating Aspergillus nidulans hydrolytic enzyme production. Biotechnol Biofuels 6:91. https://doi.org/10.1186/1754-6834-6-91
Cánovas D, Studt L, Marcos AT, Strauss J (2017) High-throughput format for the phenotyping of fungi on solid substrates. Sci Rep 7:4289. https://doi.org/10.1038/s41598-017-03598-9
Cullen PJ, Sprague GF Jr (2012) The regulation of filamentous growth in yeast. Genetics 190:23–49. https://doi.org/10.1534/genetics.111.127456
Emri T, Molnár Z, Pusztahelyi T, Pócsi I (2004) Physiological and morphological changes in autolysing Aspergillus nidulans cultures. Folia Microbiol (Praha) 49:277–284
Emri T, Molnár Z, Pusztahelyi T, Varecza Z, Pócsi I (2005) The FluG-BrlA pathway contributes to the initialisation of autolysis in submerged Aspergillus nidulans cultures. Mycol Res 109:757–763
Emri T, Molnár Z, Veres T, Pusztahelyi T, Dudás G, Pócsi I (2006) Glucose-mediated repression of autolysis and conidiogenesis in Emericella nidulans. Mycol Res 110:1172–1178. https://doi.org/10.1016/j.mycres.2006.07.006
Emri T, Molnár Z, Szilágyi M, Pócsi I (2008) Regulation of autolysis in Aspergillus nidulans. Appl Biochem Biotechnol 151:211–220. https://doi.org/10.1007/s12010-008-8174-7
Han KH (2009) Molecular genetics of Emericella nidulans sexual development. Mycobiology 37:171–182. https://doi.org/10.4489/MYCO.2009.37.3.171
Han DM, Han YJ, Lee YH, Jahng KY, Jahng SH, Chae KS (1990) Inhibitory conditions of asexual development and their application for the screening of mutants defective in sexual development. Korean J Mycol 18:225–232
Han KH, Han KY, Yu JH, Chae KS, Jahng KY, Han DM (2001) The nsdD gene encodes a putative GATA-type transcription factor necessary for sexual development of Aspergillus nidulans. Mol Microbiol 41:299–309
Han KH, Lee DB, Kim JH, Han DM (2003) Environmental factors affecting development of Aspergillus nidulans. J Microbiol 41:34–40
Hoffmann B, Wanke C, Lapaglia SK, Braus GH (2000) c-Jun and RACK1 homologues regulate a control point for sexual development in Aspergillus nidulans. Mol Microbiol 37:28–41
Jeong HY, Han DM, Jahng KY, Chae KS (2000) The rpl16a gene for ribosomal protein L16A identified from expressed sequence tags is differentially expressed during sexual development of Aspergillus nidulans. Fungal Genet Biol 31:69–78. https://doi.org/10.1006/fgbi.2000.1233
Katz ME, Bernardo SM, Cheetham BF (2008) The interaction of induction, repression and starvation in the regulation of extracellular proteases in Aspergillus nidulans: evidence for a role for CreA in the response to carbon starvation. Curr Genet 54:47–55. https://doi.org/10.1007/s00294-008-0198-6
Katz ME, Braunberger K, Yi G, Cooper S, Nonhebel HM, Gondro C (2013) A p53-like transcription factor similar to Ndt80 controls the response to nutrient stress in the filamentous fungus, Aspergillus nidulans. F1000Res 2:72. https://doi.org/10.12688/f1000research.2-72.v1
Kawasaki L, Sánchez O, Shiozaki K, Aquirre J (2002) SakA MAP kinase is involved in stress signal transduction, sexual development and spore viability in Aspergillus nidulans. Mol Microbiol 45:1153–1163
Kikuma T, Ohneda M, Arioka M, Kitamoto K (2006) Functional analysis of the ATG8 homologue Aoatg8 and role of autophagy in differentiation and germination in Aspergillus oryzae. Eukaryot Cell 5:1328–1336. https://doi.org/10.1128/EC.00024-06
Krijgsheld P, Bleichrodt R, van Veluw GJ, Wang F, Müller WH, Dijksterhuis J, Wösten HA (2013a) Development in Aspergillus. Stud Mycol 74:1–29. https://doi.org/10.3114/sim0006
Krijgsheld P, Nitsche BM, Post H, Levin AM, Müller WH, Heck AJ, Ram AF, Altelaar AF, Wösten HA (2013b) Deletion of flbA results in increased secretome complexity and reduced secretion heterogeneity in colonies of Aspergillus niger. J Proteome Res 12:1808–1819. https://doi.org/10.1021/pr301154w
Leary NO, Pembroke A, Duggan PF (1992) Improving accuracy of glucose oxidase procedure for glucose determinations on discrete analyzers. Clin Chem 38:298–302
Lee BN, Adams TH (1996) fluG and flbA function interdependently to initiate conidiophore development in Aspergillus nidulans through brlAβ activation. EMBO J 15:299–309
Levin AM, de Vries RP, Conesa A, de Bekker C, Talon M, Menke HH, van Peij NN, Wösten HA (2007) Spatial differentiation in the vegetative mycelium of Aspergillus niger. Eukaryot Cell 6:2311–2322. https://doi.org/10.1128/EC.00244-07
Matsuura S (1998) Colony pattering of Aspergillus oryzae on agar media. Mycoscience 39:379–390. https://doi.org/10.1007/BF02460898
Matsuura S (2002) Colony patterning and collective hyphal growth of filamentous fungi. Physica A 315:125–136. https://doi.org/10.1016/S0378-4371(02)01249-9
McCluskey K (2003) The fungal genetics stock center: from molds to molecules. Adv Appl Microbiol 52:245–262
Molnár Z, Mészáros E, Szilágyi Z, Rosén S, Emri T, Pócsi I (2004) Influence of fadA G203R and ΔflbA mutations on the morphology and physiology of submerged Aspergillus nidulans cultures. Appl Biochem Biotechnol 118:349–360
Molnár Z, Emri T, Zavaczki E, Pusztehelyi T, Pócsi I (2006) Effects of mutations in the GanB/RgsA G protein mediated signaling on the autolysis of Aspergillus nidulans. J Basic Microbiol 46:495–503. https://doi.org/10.1002/jobm.200610174
van Munster J, Burggraaf A, Pócsi I, Szilágyi M, Emri T, Ram A (2016) Post-genomic approaches to dissect carbon starvation responses in aspergilli. In: de Vries RP, Gelber IB, Andersen MR (eds) Aspergillus and Penicillium in the post-genomic era, Caister academic press, Norfolk, pp 89–111
Nitsche BM, Burggraaf-Van Welzen AM, Lamers G, Meyer V, Ram AFJ (2013) Autophagy promotes survival in aging submerged cultures of the filamentous fungus Aspergillus niger. Appl Microbiol Biotechnol 97:8205–8218. https://doi.org/10.1007/s00253-013-4971-1
Palecek SP, Parikh AS, Kron SJ (2002) Sensing, signalling and integrating physical processes during Saccharomyces cerevisiae invasive and filamentous growth. Microbiology 148:893–907. https://doi.org/10.1099/00221287-148-4-893
Pan X, Heitman J (1999) Cyclic AMP-dependent protein kinase regulates pseudohyphal differentiation in Saccharomyces cerevisiae. Mol Cell Biol 19:4874–4887
Pócsi I, Leiter É, Kwon NJ, Shin KS, Kwon GS, Pusztahelyi T, Emri T, Abuknesha R, Price R, Yu JH (2009) Asexual sporulation signaling regulates autolysis of Aspergillus nidulans via modulating the chitinase ChiB production. J Appl Microbiol 107:514–523. https://doi.org/10.1111/j.1365-2672.2009.04237.x
Pusztahelyi T, Pócsi I (2014) Chitinase but not N-acetyl-β-D-glucosaminidase production correlates to the biomass decline in Penicillium and Aspergillus species. Acta Microbiol Immunol Hung 61:131–143. https://doi.org/10.1556/AMicr.61.2014.2.4
Pusztahelyi T, Molnár Z, Emri T, Klement E, Miskei M, Kerékgyárto J, Balla J, Pócsi I (2006) Comparative studies of differential expression of chitinolytic enzymes encoded by chiA, chiB, chiC and nagA genes in Aspergillus nidulans. Folia Microbiol (Praha) 51:547–554
Richie DL, Fuller KK, Fortwendel J, Miley MD, McCarthy JW, Feldmesser M, Rhodes JC, Askew DS (2007) Unexpected link between metal ion deficiency and autophagy in Aspergillus fumigatus. Eukaryot Cell 6:2437–2447. https://doi.org/10.1128/EC.00224-07
Ries LN, Beattie SR, Espeso EA, Cramer RA, Goldman GH (2016) Diverse regulation of the CreA carbon catabolite repressor in Aspergillus nidulans. Genetics 203:335–352. https://doi.org/10.1534/genetics.116.187872
Shroff RA, O’Connor SM, Hynes MJ, Lockington RA, Kelly JM (1997) Null alleles of creA, the regulator of carbon catabolite repression in Aspergillus nidulans. Fungal Genet Biol 22:28–38. https://doi.org/10.1006/fgbi.1997.0989
Sipiczki M, Takeo K, Yamaguchi M, Yoshida S, Miklos I (1998) Environmentally controlled dimorphic cycle in a fission yeast. Microbiology 144:1319–1330. https://doi.org/10.1099/00221287-144-5-1319
Spitzmüller Z, Kwon NJ, Szilágyi M, Keserű J, Tóth V, Yu JH, Pócsi I, Emri T (2015) γ-Glutamyl transpeptidase (GgtA) of Aspergillus nidulans is not necessary for bulk degradation of glutathione. Arc Microbiol 197:285–297. https://doi.org/10.1007/s00203-014-1057-0
Szilágyi M, Kwon NJ, Dorogi C, Pócsi I, Yu JH, Emri T (2010) The extracellular β-1,3-endoglucanase EngA is involved in autolysis of Aspergillus nidulans. J Appl Microbiol 109:1498–1508. https://doi.org/10.1111/j.1365-2672.2010.04782.x
Szilágyi M, Kwon N-J, Bakti F, M-Hamvas M, Jámbrik K, Park HS, Pócsi I, Yu JH, Emri T (2011) Extracellular proteinase formation in carbon starving Aspergillus nidulans cultures—physiological function and regulation. J Basic Microbiol 51:625–634. https://doi.org/10.1002/jobm.201100068
Szilágyi M, Anton F, Pócsi I, Emri T (2018) Autolytic enzymes are responsible for increased melanization of carbon stressed Aspergillus nidulans cultures. J Basic Microbiol. https://doi.org/10.1002/jobm.201700545
Wang F, Krijgsheld P, Hulsman M, de Bekker C, Müller WH, Reinders M, de Vries RP, Wösten HA (2015) FluG affects secretion in colonies of Aspergillus niger. Antonie Van Leeuwenhoek 107:225–240. https://doi.org/10.1007/s10482-014-0321-2
Yamazaki H, Yamazaki D, Takaya N, Takagi M, Ohta A, Horiuchi H (2007) A chitinase gene, chiB, involved in the autolytic process of Aspergillus nidulans. Curr Genet 51:89–98. https://doi.org/10.1007/s00294-006-0109-7
Yu JH (2006) Heterotrimeric G protein signaling and RGSs in Aspergillus nidulans. J Microbiol 44:145–154
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This work was supported by the National Research, Development and Innovation Office (K112181).
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Emri, T., Vékony, V., Gila, B. et al. Autolytic hydrolases affect sexual and asexual development of Aspergillus nidulans. Folia Microbiol 63, 619–626 (2018). https://doi.org/10.1007/s12223-018-0601-8
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DOI: https://doi.org/10.1007/s12223-018-0601-8