Applied Microbiology and Biotechnology

, Volume 103, Issue 9, pp 3829–3846 | Cite as

Morphological, transcriptional, and metabolic analyses of osmotic-adapted mechanisms of the halophilic Aspergillus montevidensis ZYD4 under hypersaline conditions

  • Xiaowei Ding
  • Kaihui LiuEmail author
  • Yuxin Lu
  • Guoli Gong
Genomics, transcriptomics, proteomics


Halophilic fungi in hypersaline habitats require multiple cellular responses for high-salinity adaptation. However, the exact mechanisms behind these adaptation processes remain to be slightly known. The current study is aimed at elucidating the morphological, transcriptomic, and metabolomic changes of the halophilic fungus Aspergillus montevidensis ZYD4 under hypersaline conditions. Under these conditions, the fungus promoted conidia formation and suppressed cleistothecium development. Furthermore, the fungus differentially expressed genes (P < 0.0001) that controlled ion transport, amino acid transport and metabolism, soluble sugar accumulation, fatty acid β-oxidation, saturated fatty acid synthesis, electron transfer, and oxidative stress tolerance. Additionally, the hypersalinized mycelia widely accumulated metabolites, including amino acids, soluble sugars, saturated fatty acids, and other carbon- and nitrogen-containing compounds. The addition of metabolites—such as neohesperidin, biuret, aspartic acid, alanine, proline, and ornithine—significantly promoted the growth (P ≤ 0.05) and the morphological adaptations of A. montevidensis ZYD4 grown in hypersaline environments. Our study demonstrated that morphological shifts, ion equilibrium, carbon and nitrogen metabolism for solute accumulation, and energy production are vital to halophilic fungi so that they can build tolerance to high-salinity environments.


Transcriptome Metabolome Halophilic fungus Aspergillus montevidensis Hypersaline adaptation Carbon and nitrogen metabolism 



We are especially grateful to the editor and reviewers for their valuable comments on the manuscript. The manuscript was linguistically edited by Natalie Kaplan, Johns Hopkins University and by Long Lynette, Dartmouth College.

Funding information

This work was supported by the National Natural Science Foundation of China (no. 31100017) and by the Program of Agricultural Scientific and Technological Innovation of Shaanxi Province (2018NY-156).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Ethical approval

This article does not contain any studies performed with human participants or with animals by the author.

Supplementary material

253_2019_9705_MOESM1_ESM.pdf (671 kb)
ESM 1 (PDF 670 kb)


  1. Al-Mailem DM, Eliyas M, Radwan SS (2018) Ferric sulfate and proline enhance heavy-metal tolerance of halophilic/halotolerant soil microorganisms and their bioremediation potential for spilled-oil under multiple stresses. Front Microbiol 9:394. Google Scholar
  2. Bass RB, Strop P, Barclay M, Rees DC (2002) Crystal structure of Escherichia coli MscS, a voltage-modulated and mechanosensitive channel. Science 298(5598):1582–1587. Google Scholar
  3. Cheng C, Dong Z, Han X, Wang H, Jiang L, Sun J, Yang Y, Ma T, Shao C, Wang X, Chen Z, Fang W, Freitag NE, Huang H, Song H (2017) Thioredoxin A is essential for motility and contributes to host infection of Listeria monocytogenes via redox interactions. Front Cell Infect Microbiol 7:287. Google Scholar
  4. Chung H, Choi J, Park SY, Jeon J, Lee YH (2013) Two conidiation related Zn (II)2Cys6 transcription factor genes in the rice blast fungus. Fungal Genet Biol 61:133–141. Google Scholar
  5. Coninx L, Thoonen A, Slenders E, Morin E, Arnauts N, Op De Beeck M, Kohler A, Ruytinx J, Colpaert JV (2017) The SlZRT1 gene encodes a plasma membrane-located ZIP (Zrt-, Irt-like protein) transporter in the ectomycorrhizal fungus Suillus luteus. Front Microbiol 8:2320. Google Scholar
  6. Dunn WB, Broadhurst D, Begley P, Zelena E, Francis-McIntyre S, Anderson N, Brown M, Knowles JD, Halsall A, Haselden JN, Nicholls AW, Wilson ID, Kell DB, Goodacre R, Human Serum Metabolome (HUSERMET) Consortium (2011) Procedures for large-scale metabolic profiling of serum and plasma using gas chromatography and liquid chromatography coupled to mass spectrometry. Nat Protoc 6(7):1060–1083. Google Scholar
  7. Gil-Durán C, Rojas-Aedo JF, Medina E, Vaca I, García-Rico RO, Villagrán S, Levicán G, Chávez R (2015) The pcz1 gene, which encodes a Zn (II)2Cys6 protein, is involved in the control of growth, conidiation, and conidial germination in the filamentous fungus Penicillium roqueforti. PLoS One 10:e0120740. Google Scholar
  8. Gostinčar C, Gunde-Cimerman N (2018) Overview of oxidative stress response genes in selected halophilic fungi. Genes (Basel) 9(3):E143. Google Scholar
  9. Götz S, García-Gómez JM, Terol J, Williams TD, Nagaraj SH, Nueda MJ, Robles M, Talón M, Dopazo J, Conesa A (2008) High-throughput functional annotation and data mining with the Blast2GO suite. Nucleic Acids Res 36(10):3420–3435. Google Scholar
  10. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, Chen Z, Mauceli E, Hacohen N, Gnirke A, Rhind N, di Palma F, Birren BW, Nusbaum C, Lindblad-Toh K, Friedman N, Regev A (2011) Full-length transcriptome assembly from RNA-seq data without a reference genome. Nat Biotechnol 29(7):644–652. Google Scholar
  11. Gunde-Cimerman N, Plemenitaš A, Oren A (2018) Strategies of adaptation of microorganisms of the three domains of life to high salt concentrations. FEMS Microbiol Rev 42(3):353–375. Google Scholar
  12. Guo S, Yao Y, Zuo L, Shi W, Gao N, Xu H (2016) Enhancement of tolerance of Ganoderma lucidum to cadmium by nitric oxide. J Basic Microbiol 56(1):36–43. Google Scholar
  13. Hachicho N, Birnbaum A, Heipieper HJ (2017) Osmotic stress in colony and planktonic cells of Pseudomonas putida mt-2 revealed significant differences in adaptive response mechanisms. AMB Express 7(1):62. Google Scholar
  14. Han JM, Jeong SJ, Park MC, Kim G, Kwon NH, Kim HK, Ha SH, Ryu SH, Kim S (2012) Leucyl-tRNA synthetase is an intracellular leucine sensor for the mTORC1-signaling pathway. Cell 149(2):410–424. Google Scholar
  15. Harding T, Roger AJ, Simpson AGB (2017) Adaptations to high salt in a halophilic protist: differential expression and gene acquisitions through duplications and gene transfers. Front Microbiol 8:944. Google Scholar
  16. He Z, Zhou A, Baidoo E, He Q, Joachimiak MP, Benke P, Phan R, Mukhopadhyay A, Hemme CL, Huang K, Alm EJ, Fields MW, Wall J, Stahl D, Hazen TC, Keasling JD, Arkin AP, Zhou J (2010) Global transcriptional, physiological, and metabolite analyses of the responses of Desulfovibrio vulgaris Hildenborough to salt adaptation. Appl Environ Microbiol 76(5):1574–1586. Google Scholar
  17. He B, Ma L, Hu Z, Li H, Ai M, Long C, Zeng B (2018) Deep sequencing analysis of transcriptomes in Aspergillus oryzae in response to salinity stress. Appl Microbiol Biotechnol 102(2):897–906. Google Scholar
  18. Henry C, Bledsoe SW, Griffiths CA, Kollman A, Paul MJ, Sakr S, Lagrimini LM (2015) Differential role for trehalose metabolism in salt-stressed maize. Plant Physiol 169(2):1072–1089. Google Scholar
  19. Hilbish TJ, Koehn RK (1985) The physiological basis of natural selection at the lap locus. Evolution 39(6):1302–1317. Google Scholar
  20. Hirasawa T, Nakakura Y, Yoshikawa K, Ashitani K, Nagahisa K, Furusawa C, Katakura Y, Shimizu H, Shioya S (2006) Comparative analysis of transcriptional responses to saline stress in the laboratory and brewing strains of Saccharomyces cerevisiae with DNA microarray. Appl Microbiol Biotechnol 70(3):346–357. Google Scholar
  21. Hohmann S, Krantz M, Nordlander B (2007) Yeast osmoregulation. Methods Enzymol 428:29–45. Google Scholar
  22. Inbar E, Schlisselberg D, Suter Grotemeyer M, Rentsch D, Zilberstein D (2013) A versatile proline/alanine transporter in the unicellular pathogen Leishmania donovani regulates amino acid homoeostasis and osmotic stress responses. Biochem J 449(2):555–566. Google Scholar
  23. Jewell JL, Russell RC, Guan KL (2013) Amino acid signalling upstream of mTOR. Nat Rev Mol Cell Biol 14(3):133–139. Google Scholar
  24. Kanehisa M, Sato Y, Kawashima M, Furumichi M, Tanabe M (2016) KEGG as a reference resource for gene and protein annotation. Nucleic Acids Res 44(D1):D457–D462. Google Scholar
  25. Kim YJ, Yu YM, Maeng PJ (2017) Differential control of asexual development and sterigmatocystin biosynthesis by a novel regulator in Aspergillus nidulans. Sci Rep 7:46340. Google Scholar
  26. Kis-Papo T, Weig AR, Riley R, Peršoh D, Salamov A, Sun H, Lipzen A, Wasser SP, Rambold G, Grigoriev IV, Nevo E (2014) Genomic adaptations of the halophilic Dead Sea filamentous fungus Eurotium rubrum. Nat Commun 5:3745. Google Scholar
  27. Klähn S, Hagemann M (2011) Compatible solute biosynthesis in cyanobacteria. Environ Microbiol 13(3):551–562. Google Scholar
  28. Kogej T, Ramos J, Plemenitas A, Gunde-Cimerman N (2005) The halophilic fungus Hortaea werneckii and the halotolerant fungus Aureobasidium pullulans maintain low intracellular cation concentrations in hypersaline environments. Appl Environ Microbiol 71(11):6600–6605. Google Scholar
  29. Kogej T, Gorbushina AA, Gunde-Cimerman N (2006) Hypersaline conditions induce changes in cell-wall melanization and colony structure in a halophilic and a xerophilic black yeast species of the genus Trimmatostroma. Mycol Res 110(Pt 6):713–724. Google Scholar
  30. Kogej T, Stein M, Volkmann M, Gorbushina AA, Galinski EA, Gunde-Cimerman N (2007) Osmotic adaptation of the halophilic fungus Hortaea werneckii: role of osmolytes and melanization. Microbiology 153(Pt 12):4261–4273. Google Scholar
  31. Kralj Kuncic M, Kogej T, Drobne D, Gunde-Cimerman N (2010) Morphological response of the halophilic fungal genus Wallemia to high salinity. Appl Environ Microbiol 76(1):329–337. Google Scholar
  32. Lee JM, Gianchandani EP, Papin JA (2006) Flux balance analysis in the era of metabolomics. Brief Bioinform 7(2):140–150. Google Scholar
  33. Lee DJ, Chi YT, Kim DM, Choi SH, Lee JY, Choi GW (2014) Ectopic expression of CaRLK1 enhances hypoxia tolerance with increasing alanine production in Nicotiana spp. Plant Mol Biol 86(3):255–270. Google Scholar
  34. Lemire J, Alhasawi A, Appanna VP, Tharmalingam S, Appanna VD (2017) Metabolic defence against oxidative stress: the road less travelled so far. J Appl Microbiol 123(4):798–809. Google Scholar
  35. Liu KH, Ding XW, Narsing Rao MP, Zhang B, Zhang YG, Liu FH, Liu BB, Xiao M, Li WJ (2017a) Morphological and transcriptomic analysis reveals the osmoadaptive response of endophytic fungus Aspergillus montevidensis ZYD4 to high salt stress. Front Microbiol 8:1789. Google Scholar
  36. Liu SM, Li JX, Wu Y, Ren YN, Liu Q, Wang QY, Zhou XS, Cai MH, Zhang YX (2017b) De novo transcriptome sequencing of marine-derived Aspergillus glaucus and comparative analysis of metabolic and developmental variations in response to salt stress. Genes Genomics 39(3):317–329. Google Scholar
  37. Majee M, Maitra S, Dastidar KG, Pattnaik S, Chatterjee A, Hait NC, Das KP, Majumder AL (2004) A novel salt-tolerant L-myo-inositol-1-phosphate synthase from Porteresia coarctata (Roxb.) Tateoka, a halophytic wild rice: molecular cloning, bacterial overexpression, characterization, and functional introgression into tobacco-conferring salt tolerance phenotype. J Biol Chem 279(27):28539–28552. Google Scholar
  38. McMahon B, Gallagher ME, Mayhew SG (2005) The protein coded by the PP2216 gene of Pseudomonas putida KT2440 is an acyl-CoA dehydrogenase that oxidises only short-chain aliphatic substrates. FEMS Microbiol Lett 250(1):121–127. Google Scholar
  39. Merlino G, Barozzi A, Michoud G, Ngugi DK, Daffonchio D (2018) Microbial ecology of deep-sea hypersaline anoxic basins. FEMS Microbiol Ecol 94(7):fiy085. Google Scholar
  40. Mesbah NM, Wiegel J (2012) Life under multiple extreme conditions: diversity and physiology of the halophilic alkalithermophiles. Appl Environ Microbiol 78(12):4074–4082. Google Scholar
  41. Moriya Y, Itoh M, Okuda S, Yoshizawa AC, Kanehisa M (2007) KAAS: an automatic genome annotation and pathway reconstruction server. Nucleic Acids Res 35(Web Server):W182–W185. Google Scholar
  42. Oren A (1999) Bioenergetic aspects of halophilism. Microbiol Mol Biol Rev 63(2):334–348. Google Scholar
  43. Orino K, Lehman L, Tsuji Y, Ayaki H, Torti SV, Torti FM (2001) Ferritin and the response to oxidative stress. Biochem J 357(Pt1):241–247. Google Scholar
  44. Pandit PR, Fulekar MH, Karuna MSL (2017) Effect of salinity stress on growth, lipid productivity, fatty acid composition, and biodiesel properties in Acutodesmus obliquus and Chlorella vulgaris. Environ Sci Pollut Res Int 24(15):13437–13451. Google Scholar
  45. Plemenitaš A, Lenassi M, Konte T, Kejžar A, Zajc J, Gostinčar C, Gunde-Cimerman N (2014) Adaptation to high salt concentrations in halotolerant/halophilic fungi: a molecular perspective. Front Microbiol 5:199. Google Scholar
  46. Plemenitaš A, Konte T, Gostinčar C, Cimerman NG (2016) Transport systems in halophilic fungi. Adv Exp Med Biol 892:307–325. Google Scholar
  47. Ravishankar JP, Suryanarayanan TS, Muruganandam V (2006) Strategies for osmoregulation in the marine fungus Cirrenalia pygmea Kohl. (Hyphomycetes). Indian J Mar Sci 35(4):351–358. Google Scholar
  48. Robert H, Le Marrec C, Blanco C, Jebbar M (2000) Glycine betaine, carnitine, and choline enhance salinity tolerance and prevent the accumulation of sodium to a level inhibiting growth of Tetragenococcus halophila. Appl Environ Microbiol 66(2):509–517. Google Scholar
  49. Robinson MD, McCarthy DJ, Smyth GK (2010) edgeR: a bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26(1):139–140. Google Scholar
  50. Rodríguez-Navarro A, Benito B (2010) Sodium or potassium efflux ATPase: a fungal, bryophyte, and protozoal ATPase. Biochim Biophys Acta 1798(10):1841–1853. Google Scholar
  51. Roesser M, Müller V (2001) Osmoadaptation in bacteria and archaea: common principles and differences. Environ Microbiol 3(12):743–754. Google Scholar
  52. Saccenti E, Hoefsloot HCJ, Smilde AK, Westerhuis JA, Hendriks MMWB (2014) Reflections on univariate and multivariate analysis of metabolomics data. Metabolomics 10(3):361–374. Google Scholar
  53. Sansom FM, Tang L, Ralton JE, Saunders EC, Naderer T, McConville MJ (2013) Leishmania major methionine sulfoxide reductase A is required for resistance to oxidative stress and efficient replication in macrophages. PLoS One 8(2):e56064. Google Scholar
  54. Segev N, Hay N (2012) Hijacking leucyl-tRNA synthetase for amino acid-dependent regulation of TORC1. Mol Cell 46(1):4–6. Google Scholar
  55. Son H, Park AR, Lim JY, Lee YW (2015) Fss1 is involved in the regulation of an ENA5 homologue for sodium and lithium tolerance in Fusarium graminearum. Environ Microbiol 17(6):2048–2063. Google Scholar
  56. St John G, Brot N, Ruan J, Erdjument-Bromage H, Tempst P, Weissbach H, Nathan C (2001) Peptide methionine sulfoxide reductase from Escherichia coli and Mycobacterium tuberculosis protects bacteria against oxidative damage from reactive nitrogen intermediates. Proc Natl Acad Sci U S A 98(17):9901–9906. Google Scholar
  57. Stevenson A, Cray JA, Williams JP, Santos R, Sahay R, Neuenkirchen N, McClure CD, Grant IR, Houghton JD, Quinn JP, Timson DJ, Patil SV, Singhal RS, Antón J, Dijksterhuis J, Hocking AD, Lievens B, Rangel DE, Voytek MA, Gunde-Cimerman N, Oren A, Timmis KN, McGenity TJ, Hallsworth JE (2015) Is there a common water-activity limit for the three domains of life? ISME J 9(6):1333–1351. Google Scholar
  58. Thomas SP, Shanmugasundaram S (1991) Osmoregulatory role of alanine during salt stress in the nitrogen fixing cyanobacterium Anabaena sp. 287. Biochem Int 23(1):93–102. Google Scholar
  59. Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, Salzberg SL, Wold BJ, Pachter L (2010) Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol 28(5):511–515. Google Scholar
  60. Trygg J, Wold S (2002) Orthogonal projections to latent structures (O-PLS). J Chemom 16(3):119–128. Google Scholar
  61. Varga J, Szigeti G, Baranyi N, Kocsubé S, O'Gorman CM, Dyer PS (2014) Aspergillus: sex and recombination. Mycopathologia 178(5–6):349–362. Google Scholar
  62. Wang W, Black SS, Edwards MD, Miller S, Morrison EL, Bartlett W, Dong C, Naismith JH, Booth IR (2008) The structure of an open form of an E. coli mechanosensitive channel at 3.45 A resolution. Science 321(5893):1179–1183. Google Scholar
  63. Wendell SG, Golin-Bisello F, Wenzel S, Sobol RW, Holguin F, Freeman BA (2015) 15-Hydroxyprostaglandin dehydrogenase generation of electrophilic lipid signaling mediators from hydroxy ω-3 fatty acids. J Biol Chem 290(9):5868–5880. Google Scholar
  64. Wilson ME, Maksaev G, Haswell ES (2013) MscS-like mechanosensitive channels in plants and microbes. Biochemistry 52(34):5708–5722. Google Scholar
  65. Wu C, Zhang J, Du G, Chen J (2013) Aspartate protects Lactobacillus casei against acid stress. Appl Microbiol Biotechnol 97(9):4083–4093. Google Scholar
  66. Zajc J, Liu Y, Dai W, Yang Z, Hu J, Gostinčar C, Gunde-Cimerman N (2013) Genome and transcriptome sequencing of the halophilic fungus Wallemia ichthyophaga: haloadaptations present and absent. BMC Genomics 14:617. Google Scholar
  67. Zajc J, Kogej T, Galinski EA, Ramos J, Gunde-Cimerman N (2014) Osmoadaptation strategy of the most halophilic fungus, Wallemia ichthyophaga, growing optimally at salinities above 15% NaCl. Appl Environ Microbiol 80(1):247–256. Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.School of Food and Biological EngineeringShaanxi University of Science and TechnologyXi’anChina
  2. 2.School of Biological Science and Engineering|Shaanxi University of TechnologyHanzhong CityChina

Personalised recommendations