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Annals of Microbiology

, Volume 69, Issue 4, pp 395–405 | Cite as

Chitinolytic actinobacteria isolated from an Algerian semi-arid soil: development of an antifungal chitinase-dependent assay and GH18 chitinase gene identification

  • Meriem GasmiEmail author
  • Mahmoud Kitouni
  • Lorena Carro
  • Petar Pujic
  • Philippe Normand
  • Hasna BoubakriEmail author
Original Article
  • 184 Downloads

Abstract

The purpose of this study was to explore the microbial potential of a semi-arid sandy soil from south-central Algeria in order to isolate new chitinolytic actinobacteria. This soil is subjected to high temperatures (up to 43 °C) and has low nutrient content. Strains were isolated by plating soil suspensions on Bennett and Colloidal Chitin (CCM) medium. An initial clustering of isolates was made through BOX-PCR genetic profiling. Next, a 16S rRNA gene sequencing of representative isolates was realized. We also identified optimum physicochemical conditions for chitinolytic activity. A rapid in vitro assay based on glucose catabolic repression was developed to select isolates having a chitinase-dependent antifungal activity against two phytopathogenic fungi. Gene identification of glycosyl hydrolase family 18 (GH18) permitted us to assess the divergence of chitinase genes. Forty isolates were obtained from the semi-arid sandy soil. The molecular identification permitted us to assign them to Streptomyces or Micromonospora genera with seven possibly new bacterial species. For chitinolytic activity, 100% of isolates were able to grow and degrade colloidal chitin at pH 7 and at a temperature ranging from 30 to 40 °C. We also observed that Micromonospora strains had atypical activity patterns, with a strong chitinase activity maintained at high temperature. Finally, three strains presented an interesting chitinolytic potential to reduce fungal growth with new GH18 sequences. This study presents a new rapid method to detect antifungal chitinase-dependent activity that allowed to identify potentially new species of actinobacteria and new GH18 gene sequences.

Keywords

Actinobacteria Chitinase BOX-PCR Catabolic repression Antifungal activity Gene capture GH18 Streptomyces Micromonospora 

Notes

Acknowledgments

This work was supported by University Frères Mentouri Constantine1 (Algeria). LC thanks the Spanish government for a postdoctoral fellowship (Programa Nacional de Movilidad de Recursos Humanos del Plan Nacional de I-D+i 2008–2011). We thank Prof. A. Boulahrouf (Constantine University) for the help with this work. Thanks are expressed to France Chitin (www.france-chitine.com) for the gift of chitin. We thank Dr Feteh-el-Zahar Haichar (University of Lyon) for the technical advice and Dr Marc Bardin (INRA, Avignon) and Dr. Merouane Fateh (Constantine University) for the gift of Botrytis cinerea Bc21 and Fusarium oxysporum, respectively.

Funding

This work was funded by the Ministry of Higher Education and Scientific Research, Algeria.

Compliance with ethical standards

Conflicts of interest

The authors declare that they have no conflict of interest.

Research involving human participants and/or animals

N/A

Informed consent

N/A

Supplementary material

13213_2018_1426_MOESM1_ESM.docx (17 kb)
ESM 1 (DOCX 16 kb)
13213_2018_1426_MOESM2_ESM.pptx (1.3 mb)
ESM 2 Fig. A1 (a) Isolation of actinobacteria on MCC media. (b) Macroscopic aspect of isolates (C13, C28, C33, and C37) cultivated on ISP-2 medium. Fig. A2 Light microscopy observations on the representative strains of Streptomyces (C27) (a) and Micromonospora (C33) (b). Micromonospora is characterized by the presence of individual spores formed at the tip of branched mycelium, not found in the other strains close to Streptomyces. Fig. A3 Chitinolytic activity measured at different pH after 7 days of growth on CCM media. (#) significant difference was observed between two conditions (Wilcoxon rank sum tests, p-value < 0.05). Fig. A4 Chitinolytic activity measured at different temperatures after 7 days of growth in CCM media and sorted according their activity at 45°C. Strains on the left of the graph (C27, C39 and C40) did not grow at 45°C and strains on the right of the graph (from C1 to S. coelicolor) did not show chitinase activity at 45°C. Better score of activity was found for six strains at 30°C (C3-C4-C13-C19-C32-C34), and one strain at 45°C (C37). (#) significant difference was observed between two conditions yielding the highest activity (Wilcoxon rank sum tests, p-value < 0.05) (PPTX 1328 kb)

References

  1. Adrangi S, Faramarzi MA (2013) From bacteria to human: a journey into the world of chitinases. Biotechnol Adv 31:1786–1795.  https://doi.org/10.1016/j.biotechadv.2013.09.012 CrossRefGoogle Scholar
  2. Anne J, Vrancken K, Van Mellaert L et al (2014) Protein secretion biotechnology in gram-positive bacteria with special emphasis on Streptomyces lividans. Biochim Biophys Acta 1843:1750–1761CrossRefGoogle Scholar
  3. Aouar L, Lerat S, Ouffroukh A et al (2012) Taxonomic identification of rhizospheric actinobacteria isolated from Algerian semi-arid soil exhibiting antagonistic activities against plant fungal pathogens. Can J Plant Pathol 34:165–176.  https://doi.org/10.1080/07060661.2012.681396 CrossRefGoogle Scholar
  4. Arimori T, Kawamoto N, Shinya S et al (2013) Crystal structures of the catalytic domain of a novel glycohydrolase family 23 chitinase from Ralstonia sp. A-471 reveals a unique arrangement of the catalytic residues for inverting chitin hydrolysis. J Biol Chem 288:18696–18706CrossRefGoogle Scholar
  5. Beier S, Bertilsson S (2013) Bacterial chitin degradation—mechanisms and ecophysiological strategies. Front Microbiol 4:. doi:  https://doi.org/10.3389/fmicb.2013.00149
  6. Bentley SD, Chater KF, Cerdeño-Tárraga A-M et al (2002) Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 417:141–147.  https://doi.org/10.1038/417141a CrossRefGoogle Scholar
  7. Boubakri H, Seghezzi N, Duchateau M et al (2015) The absence of pupylation (prokaryotic ubiquitin-like protein modification) affects morphological and physiological differentiation in Streptomyces coelicolor. J Bacteriol 197:3388–3399.  https://doi.org/10.1128/JB.00591-15 CrossRefGoogle Scholar
  8. Boudemagh A, Kitouni M, Boughachiche F (2005) Isolation and molecular identification of actinomycete microflora, of some Saharian soils of south east Algeria (Biskra, EL-Oued and Ourgla) study of antifungal activity of isolated strains. J Med Mycol 15:39–44CrossRefGoogle Scholar
  9. Carro L, Spröer C, Alonso P, Trujillo ME (2012) Diversity of Micromonospora strains isolated from nitrogen fixing nodules and rhizosphere of Pisum sativum analyzed by multilocus sequence analysis. Syst Appl Microbiol 35:73–80CrossRefGoogle Scholar
  10. Carro L, Razmilic V, Nouioui I et al (2018) Hunting for cultivable Micromonospora strains in soils of the Atacama Desert. Antonie Van Leeuwenhoek 111:1375–1387.  https://doi.org/10.1007/s10482-018-1049-1 CrossRefGoogle Scholar
  11. Chun J, Lee J-H, Jung Y (2007) EzTaxon: a web-based tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences. Int J Syst Evol Microbiol 57:2259–2261CrossRefGoogle Scholar
  12. Colson S, Stephan J, Hertrich T et al (2007) Conserved cis-acting elements upstream of genes composing the chitinolytic system of Streptomycetes are dasr-responsive elements. J Mol Microbiol Biotechnol 12:60–66CrossRefGoogle Scholar
  13. Connon SA, Lester ED, Shafaat HS et al (2007) Bacterial diversity in hyperarid Atacama Desert soils: bacterial diversity in Atacama soils. J Geophys Res Biogeosci 112:n/a–n/a.  https://doi.org/10.1029/2006JG000311 CrossRefGoogle Scholar
  14. Crawford DL, Lynch JM, Whipps JM, Ousley MA (1993) Isolation and characterization of actinomycete antagonists of a fungal root pathogen. Appl Environ Microbiol 59:3899–3905Google Scholar
  15. Dahiya N, Tewari R, Hoondal GS (2006) Biotechnological aspects of chitinolytic enzymes. Appl Microbiol Biotechnol 71:773–782CrossRefGoogle Scholar
  16. Debnath R, Saikia R, Sarma RK et al (2013) Psychrotolerant antifungal Streptomyces isolated from Tawang, India and the shift in chitinase gene family. Extremophiles 17:1045–1059CrossRefGoogle Scholar
  17. Delic I, Robbins P, Westpheling J (1992) Direct repeat sequences are implicated in the regulation of two Streptomyces chitinase promoters that are subject to carbon catabolite control. Proc Natl Acad Sci U S A 89:1885–1889CrossRefGoogle Scholar
  18. Edgar RC (2004) Muscle: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797CrossRefGoogle Scholar
  19. El-Nakeeb MA, Lechevalier HA (1963) Selective isolation of aerobic actinomycetes. Appl Microbiol 11:75–77Google Scholar
  20. Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783CrossRefGoogle Scholar
  21. Fujii T, Miyashita K (1993) Multiple domain structure in a chitinase gene (chiC) of Streptomyces lividans. J Gen Microbiol 139:677–686.  https://doi.org/10.1099/00221287-139-4-677 CrossRefGoogle Scholar
  22. Fujii T, Miyashita K, Ohtomo R, Saito A (2005) DNA-binding protein involved in the regulation of chitinase production in Streptomyces lividans. Biosci Biotechnol Biochem 69:790–799.  https://doi.org/10.1271/bbb.69.790 CrossRefGoogle Scholar
  23. Gaber Y, Mekasha S, Vaaje-Kolstad G et al (2016) Characterization of a chitinase from the cellulolytic actinomycete Thermobifida fusca. Biochim Biophys Acta 1864:1253–1259.  https://doi.org/10.1016/j.bbapap.2016.04.010 CrossRefGoogle Scholar
  24. Gasmi M, Kitouni M (2017) Optimization of chitinase production by a new Streptomyces griseorubens C9 isolate using response surface methodology. Ann Microbiol 67:175–183.  https://doi.org/10.1007/s13213-016-1249-8 CrossRefGoogle Scholar
  25. Goodfellow M, Lacey J, Todd C (1987) Numerical classification of thermophilic Streptomycetes. J Gen Microbiol 133:3135–3149Google Scholar
  26. Hamid R, Khan MA, Ahmad M et al (2013) Chitinases: an update. J Pharm Bioallied Sci 5:21–29Google Scholar
  27. Hartl L, Zach S, Seidl-Seiboth V (2012) Fungal chitinases: diversity, mechanistic properties and biotechnological potential. Appl Microbiol Biotechnol 93:533–543.  https://doi.org/10.1007/s00253-011-3723-3 CrossRefGoogle Scholar
  28. Hoang K-C, Lai T-H, Lin C-S et al (2010) The chitinolytic activities of Streptomyces sp. TH-11. Int J Mol Sci 12:56–65.  https://doi.org/10.3390/ijms12010056 CrossRefGoogle Scholar
  29. Hobel CFV, Marteinsson VT, Hreggvidsson GO, Kristjansson JK (2005) Investigation of the microbial ecology of intertidal hot springs by using diversity analysis of 16S rRNA and chitinase genes. Appl Environ Microbiol 71:2771–2776.  https://doi.org/10.1128/AEM.71.5.2771-2776.2005 CrossRefGoogle Scholar
  30. Homerova D, Knirschova R, Kormanec J (2002) Response regulator chir regulates expression of chitinase gene, chic, in Streptomyces coelicolor. Folia Microbiol (Praha) 47:499–505CrossRefGoogle Scholar
  31. Hoster F, Schmitz JE, Daniel R (2005) Enrichment of chitinolytic microorganisms: isolation and characterization of a chitinase exhibiting antifungal activity against phytopathogenic fungi from a novel Streptomyces strain. Appl Microbiol Biotechnol 66:434–442.  https://doi.org/10.1007/s00253-004-1664-9 CrossRefGoogle Scholar
  32. Hsu SC, Lockwood JL (1975) Powdered chitin agar as a selective medium for enumeration of actinomycetes in water and soil. Appl Microbiol 29:422–426Google Scholar
  33. Janda JM, Abbott SL (2007) 16S rRNA gene sequencing for bacterial identification in the diagnostic laboratory: pluses, perils, and pitfalls. J Clin Microbiol 45:2761–2764.  https://doi.org/10.1128/JCM.01228-07 CrossRefGoogle Scholar
  34. Kawase T, Saito A, Sato T et al (2004) Distribution and phylogenetic analysis of family 19 chitinases in Actinobacteria. Appl Environ Microbiol 70:1135–1144CrossRefGoogle Scholar
  35. Kawase T, Yokokawa S, Saito A et al (2006) Comparison of enzymatic and antifungal properties between family 18 and 19 chitinases from S. coelicolor A3(2). Biosci Biotechnol Biochem 70:988–998.  https://doi.org/10.1271/bbb.70.988 CrossRefGoogle Scholar
  36. Kieser T, BIBB MJ, BUTTNER MJ et al (eds) (2000) Practical Streptomyces genetics. The John Innes Foundation, NorwichGoogle Scholar
  37. Kim K-J, Yang Y-J, Kim J-G (2003) Purification and characterization of chitinase from Streptomyces sp. M-20. J Biochem Mol Biol 36:185–189Google Scholar
  38. Kishino H, Miyata T, Hasegawa M (1990) Maximum likelihood inference of protein phylogeny and the origin of chloroplasts. J Mol Evol 31:151–160CrossRefGoogle Scholar
  39. Kormanec J, Sevcikova B, and Homerova D (2000) Cloning of a two-component regulatory system probably involved. In: the regulation of chitinase in Streptomyces coelicolor a3(2). Folia Microbiol (Praha). pp 397–406Google Scholar
  40. Larsen T, Petersen BO, Storgaard BG et al (2012) Characterization of a novel Salmonella typhimurium chitinase which hydrolyzes chitin, chitooligosaccharides and an n-acetyllactosamine conjugate. Glycobiology 21:426–436CrossRefGoogle Scholar
  41. Lester ED, Satomi M, Ponce A (2007) Microflora of extreme arid Atacama Desert soils. Soil Biol Biochem 39:704–708.  https://doi.org/10.1016/j.soilbio.2006.09.020 CrossRefGoogle Scholar
  42. Liao C, Rigali S, Cassani CL et al (2014) Control of chitin and N-acetylglucosamine utilization in Saccharopolyspora erythraea. Microbiology (Reading, Engl.) 160:1914–1928.  https://doi.org/10.1099/mic.0.078261-0 CrossRefGoogle Scholar
  43. Lingappa Y, Lockwood JL (1961) A chitin medium for isolation, growth and maintenance of actinomycetes. Nature 189:158–159CrossRefGoogle Scholar
  44. Mahadevan B, Crawford DL (1997) Properties of the chitinase of the antifungal biocontrol agent Streptomyces lydicus WYEC108. Enzym Microb Technol 20:489–493CrossRefGoogle Scholar
  45. Maldonado LA, Stach JEM, Ward AC et al (2008) Characterisation of Micromonosporae from aquatic environments using molecular taxonomic methods. Antonie Van Leeuwenhoek 94:289–298.  https://doi.org/10.1007/s10482-008-9244-0 CrossRefGoogle Scholar
  46. Menna P, Pereira AA, Bangel EV, Hungria M (2009) Rep-PCR of tropical rhizobia for strain fingerprinting, biodiversity appraisal and as a taxonomic and phylogenetic tool. Symbiosis 48:120–130.  https://doi.org/10.1007/BF03179991 CrossRefGoogle Scholar
  47. Miyashita K, Fujii T, Saito A (2000) Induction and repression of a Streptomyces lividans chitinase gene promoter in response to various carbon sources. Biosci Biotechnol Biochem 64:39–43CrossRefGoogle Scholar
  48. Mohagheghi A, Grohmann K, Himmel M (1986) Isolation and characterization of Acidothermus cellulolyticus gen. nov., sp. nov., a new genus of thermophilic, acidophilic, cellulolytic bacteria. Int J Syst Bacteriol 36:435–443CrossRefGoogle Scholar
  49. Mukhtar S, Zaheer A, Aiysha D et al (2017) Actinomycetes: a source of industrially important enzymes. J Proteomics Bioinform 10:316–319.  https://doi.org/10.4172/jpb.1000456 CrossRefGoogle Scholar
  50. Murthy NKS, Bleakley BH (2012) Simplified method of preparing colloidal chitin used for screening of chitinase-producing microorganisms. Internet J Microbiol 10Google Scholar
  51. Nawani NN, Kapadnis BP (2003) Chitin degrading potential of bacteria from extreme and moderate environment. Indian J Exp Biol 41:248–254Google Scholar
  52. Nguyen J, Francou F, Virolle MJ, Guerineau M (1997) Amylase and chitinase genes in Streptomyces lividans are regulated by reg1, a pleiotropic regulatory gene. J Bacteriol 179:6383–6390CrossRefGoogle Scholar
  53. Nguyen STC, Freund HL, Kasanjian J, Berlemont R (2018) Function, distribution, and annotation of characterized cellulases, xylanases, and chitinases from CAZy. Appl Microbiol Biotechnol 102:1629–1637.  https://doi.org/10.1007/s00253-018-8778-y CrossRefGoogle Scholar
  54. Ni X, Westpheling J (1997) Direct repeat sequences in the Streptomyces chitinase-63 promoter direct both glucose repression and chitin induction. Proc Natl Acad Sci USA 94:13116–13121CrossRefGoogle Scholar
  55. Pochon J, Tardieux P (1962) Techniques d’analyse en microbiologie du sol. Editions de la Tourelle, Saint-MandéGoogle Scholar
  56. Rabbind Singh A, Senthamaraikannan P, Thangavel C et al (2014) Chis histidine kinase negatively regulates the production of chitinase chic in Streptomyces peucetius. Microbiol Res 169:155–162CrossRefGoogle Scholar
  57. Rathore AS, Gupta DR (2015) Chitinases from bacteria to human: properties, applications, and future perspectives. Enzyme Res 2015:8CrossRefGoogle Scholar
  58. Rey T, Dumas B (2017) Plenty is no plague: Streptomyces symbiosis with crops. Trends Plant Sci 22:30–37.  https://doi.org/10.1016/j.tplants.2016.10.008 CrossRefGoogle Scholar
  59. Ruiz B, Chávez A, Forero A, et al (2010) Production of microbial secondary metabolites: regulation by the carbon source. In: Critical Reviews in Microbiology. pp 146–167Google Scholar
  60. Shinya S, Fukamizo T (2017) Interaction between chitosan and its related enzymes: a review. Int J Biol Macromol 104:1422–1435.  https://doi.org/10.1016/j.ijbiomac.2017.02.040 CrossRefGoogle Scholar
  61. Simmons CW, Reddy AP, D’haeseleer P (2015) Metatranscriptomic analysis of lignocellulolytic microbial communities involved in high-solids decomposition of rice straw. Biotechnol Biofuels 31:495Google Scholar
  62. Singh R, Kumar V, Kapoor V (2014) Partial purification and characterization of a heat stable α-amylase from a thermophilic actinobacteria, Streptomyces sp. MSC702 Enzyme Res 2014:106363Google Scholar
  63. Sneath PHA, Sokal RR, Freeman WH (1975) Reviewed work: numerical taxonomy. The principles and practice of numerical classification. Syst Zool 24:263–268CrossRefGoogle Scholar
  64. Subramani R, Aalbersberg W (2013) Culturable rare Actinomycetes: diversity, isolation and marine natural product discovery. Appl Microbiol Biotechnol 97:9291–9321.  https://doi.org/10.1007/s00253-013-5229-7 CrossRefGoogle Scholar
  65. Tamura K, Stecher G, Peterson D et al (2013) MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Mol Biol Evol 30:2725–2729.  https://doi.org/10.1093/molbev/mst197 CrossRefGoogle Scholar
  66. Team RC (2012) R: a language and environment for statistical computing. R Found Stat Comput Pages.Google Scholar
  67. Trujillo ME, Alonso-Vega P, Rodriguez R et al (2010) The genus Micromonospora is widespread in legume root nodules: the example of Lupinus angustifolius. ISME J 4:1265–1281CrossRefGoogle Scholar
  68. Tsujibo H, Hatano N, Okamoto T et al (1999) Synthesis of chitinase in Streptomyces thermoviolaceus is regulated by a two-component sensor-regulator system. FEMS Microbiol Lett 181:83–90CrossRefGoogle Scholar
  69. Udaya Prakash NA, Jayanthi M, Sabarinathan R et al (2010) Evolution, homology conservation, and identification of unique sequence signatures in GH19 family chitinases. J Mol Evol 70:466–478CrossRefGoogle Scholar
  70. Ueda M, Ohata K, Konishi T et al (2009) A novel goose-type lysozyme gene with chitinolytic activity from the moderately thermophilic bacterium Ralstonia sp. A-471: cloning, sequencing, and expression. Appl Microbiol Biotechnol 81:1077–1085.  https://doi.org/10.1007/s00253-008-1676-y CrossRefGoogle Scholar
  71. Veliz AE, Martínez-Hidalgo P, Hirsch MA et al (2017) Chitinase-producing bacteria and their role in biocontrol. AIMS Microbiol 3:689–705.  https://doi.org/10.3934/microbiol.2017.3.689 CrossRefGoogle Scholar
  72. Versalovic J, Schneider M, De Bruijn FJ, Lupski J (1994) Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Methods Mol Cell Biol 5:25–40Google Scholar

Copyright information

© Università degli studi di Milano 2019

Authors and Affiliations

  1. 1.Laboratoire de Génie microbiologique et applicationsUniversité des Frères Mentouri Constantine 1ConstantineAlgeria
  2. 2.Laboratoire de Biologie appliquée et SantéUniversité des Frères Mentouri Constantine 1ConstantineAlgeria
  3. 3.Ecologie MicrobienneUniversité Lyon 1, Université de Lyon, CNRS, UMR 5557Villeurbanne CedexFrance
  4. 4.Departamento de Microbiología y GenéticaUniversidad de SalamancaSalamancaSpain

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