Culturable rare actinomycetes from Indian forest soils: Molecular and physicochemical screening for biosynthetic genes
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Abstract
Background and Objectives: Rare actinomycetes are a promising source of novel metabolites of pharmaceutical importance. The current study focussed on selective isolation of specific genera of rare actinomycetes and screening the isolates for biosynthetic genes particularly polyketide synthases (PKS) and non ribosomal peptide synthetases (NRPS).
Materials and Methods: The soil samples were subjected to various pre-treatments like 1.5% phenol treatment, 0.3% chloramine T treatment, benzethonium chloride treatment, etc. and plated on selective media supplemented with specific antibiotics targeting rare genera of actinomycetes. The putative rare actinomycete isolates were screened for bioactivity using agar cross streak method and agar well diffusion method. The ability of the isolates to produce anti-quorum sensing compounds was tested against Serratia marcescens. The isolates were also screened for the presence of biosynthetic gene clusters associated with PKS-I, PKS-II and NRPS pathways using the degenerate primer sets K1F-M6R, KSα/KSβ and A3F-A7R, respectively. The expression of these gene clusters was tracked by physicochemical screening of the extracts of isolates using spectroscopic and chromatographic techniques.
Results: In this study, 1.5% phenol treatment was found to be the most promising followed by heat treatment and chloramine treatment. Our studies showed that ISP5 agar was the best for isolation of rare genera followed by ISP7, Starch Caesin agar and ISP2 supplemented with antibiotics like gentamicin, nalidixic acid and streptomycin. Micromonospora was the most abundant genus followed by Dactylosporangium. Actinomadura, Nocardiopsis and Actinoplanes were almost equal in number. Primary screening showed that 92% of the isolates were active against one of the test organisms. Thirty seven isolates were found to produce anti-quorum sensing (QS) compounds. NRPS sequences were detected in thirty nine isolates (42.8%), whereas PKS-I and PKS-II sequences were detected in seventeen and twenty eight strains (18.6% and 30.7%), respectively.
Conclusion: Nine type I and type II polyketide-producing isolates as well as six peptide-producing isolates were found. The peptide extract of isolate KCR3 and a polyketide extract of isolate NCD10 were found to possess anti-tumor activity exhibiting an IC50 value of 3 μg/ml and 2.5 μg/ml against HeLa cells.
Metsä-Ketelä M, Halo L, Munukka E, Hakala J, Mäntsälä P, Ylihonko K. Molecular evolution of aromatic polyketides and comparative sequence analysis of polyketide ketosynthase and 16S ribosomal DNA genes from various Streptomyces species. Appl Environ Microbiol 2002; 68:4472-4479.
Mangamuri UK, Muvva V, Poda S, Kamma S. Isolation, identification and molecular characterization of rare actinomycetes from mangrove ecosystem of Nizampatnam. Mal J Microbiol 2012; 8:83-91.
Qin S, Li J, Chen HH, Zhao GZ, Zhu WY, Jiang CL, et al. Isolation, diversity, and antimicrobial activity of rare actinobacteria from medicinal plants of tropical rain forests in Xishuangbanna, China. Appl Environ Microbiol 2009; 75:6176-6186.
Okudoh VI, Wallis FM. Antimicrobial activity of rare actinomycetes isolated from natural habitats in KwaZulu-Natal, South Africa. S Afr J Sci 2007; 103(5-6):216-222.
Gontang EA, Gaudêncio SP, Fenical W, Jensen PR. Sequence-based analysis of secondary-metabolite biosynthesis in marine actinobacteria. Appl Environ Microbiol 2010; 76:2487-2499.
Ayuso-Sacido A, Genilloud O. New PCR primers for the screening of NRPS and PKS-I systems in actinomycetes: detection and distribution of these biosynthetic gene sequences in major taxonomic groups. Microb Ecol 2005; 49:10-24.
Fguira LF, Bejar S, Mellouli L. Isolation and screening of Streptomyces from soil of Tunisian oases ecosystem for nonpolyenic antifungal metabolites. Afr J Biotechnol 2012; 11:7512.-7519.
Taddei A, Valderrama M, Giarrizzo J, Rey M, Castelli C. Chemical screening: A simple approach to visualizing Streptomyces diversity for drug discovery and further research. Res Microbiol 2006; 157:291-297.
Hayakawa M. Studies on the isolation and distribution of rare Actinomycetes in soil. Actinomycetologica 2008; 22: 12-19.
Hayakawa M, Nonomura H. Efficacy of artificial humic acid as a selective nutrient in HV agar used for the isolation of soil actinomycetes. J Fermen Tech 1987; 65:609-616.
Shirling ET, Gottlieb D. Methods for characterization of Streptomyces species1. Int J Syst Evol Microbiol 1966; 16:313-340.
Becker B, Lechevalier MP, Lechevalier HA. Chemical composition of cell-wall preparations from strains of various form-genera of aerobic actinomycetes. Appl Microbiol 1965; 13:236-243.
Staneck JL, Roberts GD. Simplified approach to identification of aerobic actinomycetes by thin-layer chromatography. App Microbiol 1974; 28:226-231.
Kiska DL, Hicks K, Pettit DJ. Identification of medically relevant Nocardia species with an abbreviated battery of tests. J Clin Microbiol 2002; 40:1346-1351.
Peela S, Kurada VB, Terli R. Studies on antagonistic marine actinomycetes from the Bay of Bengal. World J Microbiol Biotechnol 2005; 21:583-585.
Grammer A. Antibiotic sensitivity and assay test. J Microbiol Methods 1976; 235.
Kanagasabhapathy M, Yamazaki G, Ishida A, Sasaki H, Nagata S. Presence of quorum-sensing inhibitor-like compounds from bacteria isolated from the brown alga Colpomenia sinuosa. Lett Appl Microbiol 2009; 49:573-579.
Metsä-Ketelä M, Salo V, Halo L, Hautala A, Hakala J, Mäntsälä P, et al. An efficient approach for screening minimal PKS genes from Streptomyces. FEMS Microbiol Lett 1999; 180:1-6.
Kieser T, Bibb MJ, Buttner M, Chater KF, Hopwood DA. Practical Streptomyces genetics, The John Innes Foundation; 2000.
Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 2004; 32:1792-1797.
Dereeper A, Guignon V, Blanc G, Audic S, Buffet S, Chevenet F, et al. Phylogeny. fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Res 2008; 36(Web Server issue):W465-469.
Bundale SB, Begde DN, Nashikkar NN, Kadam TA, Upadhyay AA. Isolation of aromatic polyketide producing soil Streptomyces using combinatorial screening strategies. OALib J 2014;1:1-16.
Hamedi J, Imanparast S, Mohammadipanah F. Molecular, chemical and biological screening of soil actinomycete isolates in seeking bioactive peptide metabolites. Iran J Microbiol 2015; 7:23-30.
Begde DN, Bundale SB, Mashitha PM, Rudra JA, Nashikkar NA, Upadhyay AA. Immunomodulatory efficacy of nisin—a bacterial lantibiotic peptide. J Pept Sci 2011; 17:438-444.
Baltz RH. Renaissance in antibacterial discovery from actinomycetes. Curr Opin Pharmacol 2008; 8: 557-563.
Preobrazhenskaia TP, Lavrova NV, Ukholina RS, Nechaeva NP. Isolation of new species of the genus Actinomadura on selective media with streptomycin and bruneomycin. Antibiotiki 1975; 20:404-409.
Kim BY, Kshetrimayum JD, Goodfellow M. Detection, selective isolation and characterisation of Dactylosporangium strains from diverse environmental samples. Syst Appl Microbiol 2011; 34:606-616.
Hayakawa M, Sadakata T, Kajiura T, Nonomura H. New methods for the highly selective isolation of Micromonospora and Microbispora from soil. J Ferment Bioengineer 1991; 72:320-326.
Istianto Y, Koesoemowidodo RS, Watanabe Y, Pranamuda H, Marwoto B. Application of phenol pretreatment for the isolation of rare Actinomycetes from Indonesian soil. Microbiol Indones 2012; 6:42.
Kavitha A, Vijayalakshmi M, Sudhakar P, Narasimha G. Screening of actinomycetes strains for the production of antifungal metabolites. Afr J Microbiol Res 2010; 4: 027-032.
Jose PA, Jebakumar SR. Phylogenetic diversity of actinomycetes cultured from coastal multipond solar saltern in Tuticorin, India. Aquat Biosyst 2012; 8:23.
Ara I, Wathnani HA, Kudo T. Population, morphological and chemotaxonomical characterization of diverse rare actinomycetes in the mangrove and medicinal plant rhizosphere. Afr J Microbiol Res 2013; 7:1480-1488.
Velho-Pereira S, Kamat NM. Actinobacteriological research in India. Indian J Exp Biol 2013; 51: 573-596.
Lee LH, Zainal N, Azman AS, Eng SK, Goh BH, Yin WF, et al. Diversity and antimicrobial activities of actinobacteria isolated from tropical mangrove sediments in Malaysia. Scientific World J 2014; doi: 10.1155/2014/698178.
Yuan M, Yu Y, Li HR, Dong N, Zhang XH. Phylogenetic diversity and biological activity of actinobacteria isolated from the Chukchi Shelf marine sediments in the Arctic Ocean. Mar Drug 2014; 12:1281-1297.
Reza Dehnad A, Yeganeh LP, Bakhshi R, Mokhtarzadeh A, Soofiani S, Monadi AR, et al. Investigation of antibacterial activity of Streptomycetes isolates from soil samples, west of Iran. Afr J Microbiol Res 2010; 4:1685-93.
McLean RJ, Pierson LS, Fuqua C. A simple screening protocol for the identification of quorum signal antagonists. J Microbiol Methods 2004; 58(3):351-360.
Pathom-Aree W, Stach JE, Ward AC, Horikoshi K, Bull AT, Goodfellow M. Diversity of actinomycetes isolated from Challenger Deep sediment (10,898 m) from the Mariana Trench. Extremophiles 2006; 10:181-189.
Wood SA, Kirby BM, Goodwin CM, Le Roes M, Meyers PR. PCR screening reveals unexpected antibiotic biosynthetic potential in Amycolatopsis sp. strain UM16. J Appl Microbiol 2007; 102:245-253.
Finking R, Marahiel MA. Biosynthesis of nonribosomal peptides. Annu Rev Microbiol 2004; 58:453-488.
Wang H, Fewer DP, Holm L, Rouhiainen L, Sivonen K. Atlas of nonribosomal peptide and polyketide biosynthetic pathways reveals common occurrence of nonmodular enzymes. Proc Natl Acad Sci U S A 2014; 111:9259-9264.
Trefzer A, Blanco G, Remsing L, Künzel E, Rix U, Lipata F, et al. Rationally designed glycosylated premithramycins: hybrid aromatic polyketides using genes from three different biosynthetic pathways. J Am Chem Soc 2002; 124:6056-6062.
Maleki H, Mashinchian O. Characterization of Streptomyces isolates with UV, FTIR spectroscopy and HPLC analyses. Bioimpacts 2011; 1:47-52.
Ilić SB, Konstantinović SS, Todorović ZB. UV/VIS analysis and antimicrobial activity of Streptomyces isolates. Facta universitatis series: Med Biol 2005; 12:44-46.
Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 1991;173:697-703.
Shearer MC. Methods for the isolation of non-sptreptomycete actinomycetes. Dev Ind Microbiol 1987; 28:91-97.
Nonomura H, Takagi S. Distribution of actinoplanetes in soils of Japan. J Fermen technol 1977; 55:423-428.
Küster E, Williams ST. Selection of media for isolation of streptomycetes. Nature 1964; 202:928-929.
Williams ST, Davies FL. Use of antibiotics for selective isolation and enumeration of actinomycetes in soil. J Gen Microbiol 1965; 38:251-261.
D'costa VM, McGrann KM, Hughes DW, Wright GD. Sampling the antibiotic resistome. Science 2006; 311:374-377.
Hayakawa M, Momose Y, Yamazaki T, Nonomura H. A method for the selective isolation of Microtetraspora glauca and related four-spored actinomycetes from soil. J Appl Microbiol 1996; 80:375-386.
Labeda DP, Kroppenstedt RM. Stackebrandtia nassauensis gen. nov., sp. nov. and emended description of the family Glycomycetaceae. Int J Syst Evol Microbiol 2005; 55:1687-1691.
| Files | ||
| Issue | Vol 10 No 2 (2018) | |
| Section | Original Article(s) | |
| Keywords | ||
| Rare actinomycetes Polyketide synthases Non ribosomal peptide synthetases | ||
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