Sains Malaysiana 53(11)(2024): 3761-3770

http://doi.org/10.17576/jsm-2024-5311-18

Pemencilan, Pencirian dan Afiliasi Filogenetik Endofit Streptomyces sp. Bioaktif daripada Pokok yang Mempunyai Nilai Perubatan

(Isolation, Characterisation and Phylogenetic Affiliation of Bioactive Endophytic Streptomyces sp. Associated with Medicinal Plants)

NURUL ‘IZZAH MOHD SARMIN^1,2 & NORAZIAH MOHAMAD ZIN^3,*

¹Centre of Preclinical Science Studies, Universiti Teknologi MARA, Sungai Buloh Campus, Jalan Hospital, 47000 Selangor, Malaysia
²Atta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA, Selangor Branch, 42300 Selangor, Malaysia
³Centre for Diagnostic, Therapeutic and Investigative Studies, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, 50300 Kuala Lumpur, Malaysia

Diserahkan: 29 Ogos 2023/Diterima: 6 September 2024

Abstrak

Kepelbagaian biologi tumbuhan di hutan Malaysia menyediakan persekitaran yang sesuai untuk pemencilan Streptomyces endofit. Strain novel Streptomyces merupakan sumber berpotensi dalam menghasilkan sebatian farmaseutikal bioaktif yang boleh dibangunkan sebagai ubatan baharu. Kajian ini bertujuan untuk memencilkan Streptomyces endofit daripada tumbuhan ubatan yang berbeza di Hutan Simpan Bangi, pengenalpastian, pencirian dan analisis filogenetik pencilan tersebut. Streptomyces endofit dipencilkan menggunakan kaedah sterilisasi permukaan dan pencilan ini seterusnya dikenal pasti menggunakan pemerhatian morfologi. Streptomyces endofit yang dipencilkan telah dikelaskan ke dalam kumpulan berdasarkan warna miselium aerial. Analisis jujukan gen 16S rRNA telah dijalankan untuk mentakrifkan hubungan filogenetik antara spesies yang berkait rapat dan juga antara strain yang tergolong dalam satu spesies. Semua pencilan kemudiannya diuji untuk aktiviti antimikrob. Tiga Streptomyces endofit iaitu SUK 8, SUK 10 dan SUK 15 telah berjaya dipencilkan daripada tumbuhan ubatan yang berbeza. Ketiga-tiga pencilan tersebut dikelaskan dalam kumpulan siri kelabu berdasarkan warna miselium aerial. Siri kelabu ini juga membentuk kelompok filogenetik yang sama berdasarkan data jujukan gen 16S rRNA. Berdasarkan analisis filogenetik jujukan gen 16S rRNA, semua pencilan dikelaskan sebagai Streptomyces eurythermus ATCC 14975^T (persamaan jujukan gen 98.5%). Pencilan SUK 8, SUK 10 dan SUK 15 juga menunjukkan corak aktiviti biologi yang serupa yang berupaya merencat sehingga 100% organisma patogen yang sama. Keputusan ini mengesahkan bahawa terdapat korelasi yang baik antara kepelbagaian fenotip antimikrob dan gen 16S rRNA. Kesimpulannya, metabolit aktif yang berpotensi daripada Streptomyces endofit boleh dijangka daripada data taksonomi yang baik.

Kata kunci: Endofit; kajian filogenetik; Streptomyces; tumbuhan ubatan; 16S Rrna

Abstract

The high biodiversity of plant species in Malaysian forests provides a suitable environment for the isolation of endophytic Streptomyces. Novel strains of Streptomyces are potential sources for producing bioactive pharmaceutical compounds that can be developed as new drug candidates. This study aims to isolate the endophytic Streptomyces from different medicinal plants in the Bangi Reserve Forest before identification, characterisation, and phylogenetic analysis of the isolates. The endophytic actinomycetes were isolated using surface-sterilization method and further identified through morphological observation.  The isolated endophytic Streptomyces were classified based on the colour of aerial mycelium. 16S rRNA gene sequence analysis was done to define the phylogenetic relationships among closely related species and strains belonging to a species. All isolates were then tested for their antimicrobial activities. Three endophytic Streptomyces, SUK 8, SUK 10 and SUK 15 were successfully isolated from different medicinal plants. These three isolates were classified into a grey series group based on the colour of aerial mycelium. The grey series also formed the same phylogenetic clade based on the 16S rRNA sequence data. Phylogenetic analysis of 16S rRNA gene sequences showed all three isolates were classified as Streptomyces eurythermus ATCC 14975^T (gene sequence similarity 98.5%). Isolates of SUK 8, SUK 10 and SUK 15 also showed similar patterns of biological activities with inhibition of up to 100% of the same pathogenic organisms.  These findings proved a good correlation between the diversity of the antimicrobial phenotype and the 16S rRNA gene. In conclusion, the potential of active metabolites from endophytic Streptomyces can be expected from a good source of taxonomy data.

Keywords: Endophytes; medicinal plants; phylogenetic study; Streptomyces; 16S rRNA

RUJUKAN

Abdel-Razek, A.S., El-Naggar, M.E., Allam, A., Morsy, O.M. & Othman, S.I. 2020. Microbial natural products in drug discovery. Processes 8: 470.

Antony-Babu, S. & Goodfellow, M. 2008. Biosystematics of alkaliphilic streptomycetes isolated from seven locations across a beach and dune sand system. Antonie van Leeuwenhoek 94(4): 581-591.

Atalan, E., Manfio, G.P., Ward, A.C., Kroppenstedt, R.M. & Goodfellow, M. 2000. Biosystematic studies on novel streptomycetes from soil. Antonie van Leeuwenhoek 77(4): 337-353.

Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D.J. 1997. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Research 25: 3389-3444.

Arasu, M.V., Duraipandiyan, V., Agastian, P. & Ignacimuthu, S. 2009. In vitro antimicrobial activity of Streptomyces spp. ERI-3 isolated from Western Ghats rock soil (India). Journal de Mycologie Médicale19: 22-28.

Azerang, P. & Sardari, S. 2017. Bioactive compound produced from Actinomycetes-Streptomyces. Chemistry 151: 1507-1523.

Bubici, G. 2018. Streptomyces spp. as biocontrol agents against Fusarium species. CAB Rev. 13: 50. 

Castillo, U.F., Browne, L., Strobel, G., Hess, W.M., Ezra, S., Pacheco, G. & Ezra, D. 2007. Biologically active endophytic streptomycetes from Nothofagus spp. and other plants in Patagonia. Microbial Ecology 53(1): 12-19.

Coombs, J.T. & Franco, C.M.M. 2003. Isolation and identification of actinobacteria from surface-sterilized wheat roots. Environmental Microbiology 69(9): 5603-5608.

Chang, T.L., Huang, T.W., Wang, Y.X., Liu, C.P., Kirby, R., Chu, C.M. & Huang, C.H. 2021. An actinobacterial isolate, Streptomyces sp. YX44, produces broad-spectrum antibiotics that strongly inhibit Staphylococcus aureus. Microorganisms 9: 630. 

Chun, J., Lee, J.H., Jung, Y., Kim, M., Kim, S., Kim. B.K. & Lim, Y.W. 2007. EzTaxon: A web-based tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences. International Journal of Systematic and Evolutionary Microbiology 57: 2259-2261.

Donald, L., Pipite, A., Subramani, R., Owen, J., Keyzers, R.A. & Taufa, T. 2022. Streptomyces: Still the biggest producer of new natural secondary metabolites, a current perspective. Microbiology Research 13(3): 418-465.

Felsenstein, J. 1985. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39: 783-791.

Fiedler, H.P., Bruntner, C., Riedlinger, J., Bull, A.T., Knutsen, G., Goodfellow, M., Jones, A., Maldonado, L., Pathom-aree, W., Beil, W., Schneider, K., Keller, S. & Sussmuth, R.D. 2008. Proximicin A, B and C, novel aminofuran antibiotic and anticancer compounds isolated from marine strains of the actinomycete Verrucosispora. The Journal of Antibiotics 61: 158-163.

Fitch, W.M. 1971. Towards defining the course of evolution: Minimum change for a specific tree topology. Systematic Zoology 20: 406-416.

Genilloud, O. 2017. Actinomycetes: Still a source of novel antibiotics. Natural Product Reports 34: 1203-1232.

Gevers, D., Cohan, F.M., Lawrence, J.G., Spratt, B.G., Coenye, T., Feil, E.J., Stackebrandt, E., Van de Peer, Y., Vandamme, P. & Thompson, F.L. 2005. Re-evaluating prokaryotic species. Nature Reviews Microbiology 3(9): 733-739.

Ghadin, N., Zin, N.M., Sabaratnam, V., Badya, N., Basri, D.F., Lian, H.H. & Sidik, N.M. 2008. Isolation and identification of novel endophytic Streptomyces SUK 06 with antimicrobial activity from Malaysian plant. Asian Journal of Plant Science 7(2): 189-194.

Goodfellow, M., Kumar, Y., Labeda, D.P. & Sembiring, L. 2007. The Streptomyces violaceusniger clade: A home for streptomycetes with rugose ornamented spores. Antonie van Leeuwenhoek 92: 173-199.

Harir, M., Bendif, H., Bellahcene, M., Fortas, Z. & Pogni, R. 2018. Streptomyces secondary metabolites. Basic Biology and Applications of Actinobacteria 6: 99-122. 

Hou, B.C., Wang, E.T., Li, Y., Jia, R.Z., Chen, W.F., Man, C.X., Sui, X.H. & Chen, W.X. 2009. Rhizobial resource associated with epidemic legumes in Tibet. Microbial Ecology 57: 69-81.

Jensen, P.R., Williams, P.G., Oh, D.C., Zeigler, L. & Fenical, W. 2007. Species-specific secondary metabolite production in marine actinomycetes of the genus Salinispora. Applied and Environmental Microbiology 73(4): 1146-1152.

Kelly, K.L. 1964. Inter-society colour council-national bureau of standards color-name charts illustrated with centroid colors.  Washington: U.S. Govt. Print. Off.

Kim, M., Na, H., Park, S.C., Jeon, Y.S., Lee, J.H, Yi, H., Won, S. & Chun, J. 2012. Introducing EzTaxon-e: A prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. International Journal of Systematic and Evolutionary Microbiology 62: 716-721.

Lapaz, M.I., Cisneros, E.J., Pianzzola, M.J. & Francis, I.M. 2019. Exploring the exceptional properties of Streptomyces: A hands-on discovery of natural products. The American Biology Teacher 81: 658-664.

Majewski, J. & Cohan, F.M. 1999. DNA sequence similarity requirements for interspecific recombination in Bacillus. Genetics 153(4): 1525-1533.

Nicault, M., Zaiter, A., Dumarcay, S., Chaimbault, P., Gelhaye, E., Leblond, P. & Bontemps, C. 2021. Elicitation of antimicrobial active compounds by streptomyces-fungus co-cultures. Microorganisms 9: 178.

Sarmin, N.I.M., Tan, G.Y.A., Franco, C.M.M., Edrada-Ebel, R., Latip, J. & Zin, N.M. 2013. Streptomyces kebangsaanensis sp. nov. an endophytic actinomycete isolated from a Malaysian ethnomedicinal plant, that produces phenazine-1-carboxylic acid. International Journal of Systematic and Evolutionary Microbiology 63: 3733-3738.

Payne, G., Ward, A.C. & Goodfellow, M. 2001. The Streptomyces clavuligerusclade: A home for clavulanic acid producing streptomycetes. The 12th International Symposium on the Biology of Actinomycetes (ISBA), 5-9 August, Vancouver, British Columbia, Canada.

Qin, S., Li, J., Chen, H.H., Zhao, G.Z. & Zhu, W.Y. 2009. Isolation, diversity and antimicrobial activity of rare actinobacteria from medicinal plants of tropical rain forests in Xishuangbanna, China. Applied Environmental Microbiology 75: 6176-6186.

Saitou, N. & Nei, M. 1987. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4: 406-425.

Satheeja, S.V. & Jebakumar, S.R.D. 2011. Phylogenetic analysis and antimicrobial activities of Streptomyces isolates from mangrove sediment. Journal of Basic Microbiology 51(1): 71-79.

Sheil, D. 1999. Tropical forest diversity, environmental change and species augmentation: After the intermediate disturbance hypothesis. Journal of Vegetation Science 10: 851-860.

Shirling, E.B. & Gottlieb, D. 1966. Methods for characterization of Streptomyces species. International Journal of Systematic Bacteriology 16: 313-340.

Stackebrandt, E. & Goebel, B.M. 1994. Taxonomic note: A place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. International  Journal of Systematic Bacteriology 44: 846-849.

Stackebrandt, E., Frederiksen, W., Garrity, G.M., Grimont, P.A.D., Kämpter, P., Maiden, M.C.J., Nesme, X., Rosselló-Mora, R., Swings, J., Trüper, H.G., Vauterin, L., Ward, A.C. & Whitman, W.B. 2002. Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology. International Journal of Systematic and Evolutionary Microbiology 52(Pt 3): 1043-1047.

Staley, J.T. & Gosink, J.J. 1999. Poles apart: Biodiversity and biogeography of sea ice bacteria. Annual Review of Microbiology 53: 189-215.

Strobel, G., Daisy, B., Castillo, U. & Harper, J. 2004. Natural products from endophytic microorganisms. Journal of Natural Products 67: 257-268.

Tamura, K., Dudley, J., Nei, M. & Kumar, S. 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24: 1596-1599.

Tan, G.Y.A., Robinson, S., Lacey, E., Brown, R., Kim, W. & Goodfellow, M. 2007. Amycolatopsis regifaucium sp. nov., a novel actinomycete that produces kigamicins. International Journal of Systematic and Evolutionary Microbiology 57(11): 2562-2567.

Thompson, J.D., Higgins, D.G. & Gibson, T.J. 1994. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: 4673-4680.

Verma, V.C., Gond, S.K., Kumar, A., Mishra, A., Kharwar, R.N. & Gange, A.C. 2009. Endophytic actinomycetes from Azadirachta indica A. Juss.: Isolation, diversity, and anti-microbial activity. Microb. Ecol. 57: 749-756.

Ward, A.C. & Allenby, N.E.E. 2018. Genome mining for the search and discovery of bioactive compounds: The Streptomyces paradigm. FEMS Microbiology Letters 365: fny240. 

Ward, A.C. & Goodfellow, M. 2004. Phylogeny and functionality: Taxonomy as a roadmap to genes. In Microbial Diversity and Bioprospecting, edited by Bull, A.T. Washington: ASM Press. pp. 288-313.

Xia, H., Li, X., Li, Z., Zhan, X., Mao, X. & Li, Y. 2020. The application of regulatory cascades in Streptomyces: Yield enhancement and metabolite mining. Frontiers in Microbiology 11: 406.

Zin, N.M., Nurul, I.M.S., Norazli, G., Dayang, F.B., Nik, M.S. & Strobel, G. 2007. Bioactive endophytic streptomyces from the Malay Peninsula. FEMS Microbiology Letters 274(1): 83-88.

*Pengarang untuk surat-menyurat; email: noraziah.zin@ukm.edu.my