LV thạc sỹ_Biodiversity and antibiotic activity of actinomycetes isolated from cat ba island vietnam

CONTENTS Acknowledgements i Abbreviations ii List of figures iii List of tables iv Abstract .1 Tóm tắt Foreword Chapter Introduction 1.1 Antibiotic 1.1.1 General introduction 1.1.2 History of the development of antibiotics 1.1.3 Classification of antibiotics .9 1.1.4 Anti-tumor antibiotics 13 1.1.5 The need of developing new antibiotics 14 1.2 Actinomycetes .14 1.2.1 General characteristics 14 1.2.2 Actinomycetes and secondary metabolites 16 1.3 Objectives of the study 16 Chapter Materials and methods .18 2.1 Work flow 18 2.2 Methods 19 2.2.1 Isolation of actinomycetes .19 2.2.2 Targeted microorganisms 21 2.2.3 Screening antibiotic producing actinomycetes .21 2.2.4 Ethyl-acetate extraction 22 2.2.5 Chromatography analyses of antibiotics 22 2.2.6 Screening for cytotoxicity 24 2.2.7 Taxonomical identification of actinomycete isolates .25 Chapter Results and discussion 28 3.1 Biodiversity of the actinomycete isolated from Catba island 28 3.2 Screening for antibiotic producing actinomycetes 29 3.3 Chromatography analyses of crude extracts of the selected strains 31 3.3.1 Thin Layer Chromatography (TLC) 31 3.3.2 High – Performance Liquid Chromatography (HPLC) 32 3.4 Primary study on cytotoxicity activity 33 3.4.1 pH-dependent color change by the selected strains .33 3.4.2 Cytotoxicity assay against human cell lines 33 3.5 Identification of the actinomycete isolates .34 3.5.1 Morphology-based identification for the Streptomyces isolates .35 3.5.2 16S rDNA sequence-based identification for the non-Streptomyces isolates 37 Conclusion and Suggestion 38 Bibliography 40 Appendix Culture media 46 Appendix The 16S rDNA sequence ………………………………………… ……46 ACKNOWLEDGMENT I first and foremost wish to express gratitude to my parents for their support, encouragement and love to me The success of this thesis can be attributed to the extensive support and assistance from my advisors, Dr … I also would like to thank all of my friends in master class and in Hanoi for their friendship and help during my stay in Hanoi … ABBREVIATIONS DNA – Deoxyribonucleic acid DNR – Daunorumycin ELISA – Enzyme linked immunosorbent assay Hep-G2 – Hepatocellular carcinoma HPLC – high performance liquid chromatography HV agar – Humic acid & vitamin agar IDA – Idarumycin Lu – Lung cancer PCR – Polymerase chain reaction RD – Rhabdosarcoma RNA – Ribonucleic acid TLC – thin - layer chromatography UNESCO – United Nations Educational, Scientific and Cultural Organization UV – Ultraviolet YS – Yeast extract – Starch agar LIST OF FIGURES Figure Title Page Figure Discovery of important antibiotics and other natural products over the years Figure Evolution of penicillin G Figure Chemical structures of daunorumycin and idarumycin .13 Figure Catba national park 19 Figure Evaluation of antimicrobial activity of the actinomycete isolates 30 Figure Analyses of crude extracts of the selected actinomycete strains via TLC 31 Figure HPLC analysis of crude extract from actinomycetes 32 Figure Color change by strains A1018 and A1073 depending on pH in the medium 33 Figure Colony morphology of the representative Streptomycete strains … 35 Figure 10 Spore-bearing aerial hyphae of the representative Streptomyces strains… 36 Figure 11Neighbor-joining tree of 16S rDNA partial sequences showing phylogenetic positions of the actinomycete strains in the relationship to type strains of the genus Nonomuraea 37 LIST OF TABLES Chapter Table 1.1 Grouping of antibiotics bases on their chemical structures 10 Chapter Table 2.1 Condition for HPLC analyses of the standards 23 Chapter Table 3.1 Taxonomical grouping of the actinomycete isolates 28 Table 3.2 Antimicrobial activity of the 17 selected actinomycete strains 30 Table 3.3 Toxicity assay against three human cell lines 34 ABSTRACT In this study, a total of 424 actinomycete strains isolated from soil and litter samples on Catba island (Haiphong, Vietnam) were subjected to a screening for the inhibitory activities against microorganisms, including bacteria (Micrococcus luteus, and Escherichia coli) and eukaria (Candida albicans and Fusarium oxysporium) Through two screening steps, 17 strains were selected for their inhibitory activity against one or more target microorganisms Crude extracts in ethyl acetate from culturing media of the selected strains were analyzed via thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC), in which chloramphenicol, kitasamycin, erythromycin and raw extract of anthracycline were used as standards The obtained results showed that antibiotic substances produced by the selected strains could not be able to classify in any group of the analyzed standards, except the strain A396 which appeared to produce chloramphenicol-like antibiotic Besides that, several human tumor cell lines have also been used for testing the inhibitory effects The results showed that among the 17 selected strains, strains (A1018, A1022 and A1073) exhibited cytotoxicity against all three cell lines including hepatocellular carcinoma, lung cancer, and rhabdosarcoma Taxonomical studies based on the morphology and 16S rDNA gene sequencing indicated that the collection of actinomycetes isolated from Catba island contained mainly Streptomyces species (about 70%) and the group of rare actinomycetes (non-Streptomyces) which made of 30% of the collection was dominated by Micromonospora, Nonomureae and Nocardia genera Of the 17 selected strains with highest antimicrobial activity, ten strains affiliated to the genus Streptomyces (as based on morphology) and seven strains to the genus Nonomuraea (as based on 16S rDNA sequence analyses) The strains selected in this study could serve as valuable sources for discovering new antibiotic substances in Vietnam … 10 activity (especially against eukaryotes) and cytotoxicity activity against human cells Such kind of correlation however has not yet been elucidated in previous studies 3.5 Identification of the actinomycete isolates As mentioned in 3.1 (Biodiversity of the actinomycete isolated from Catba island), the 424 collected isolates were classified into Streptomyces (about 70%) and non-Streptomyces (rare actinomycetes) groups The information of 17 selected isolates with the highest antimicrobial activity were shown more detail bellow 3.5.1 Morphology-based identification for the Streptomyces isolates Ten strains (A390, A410, A427, A1018, A1022, A1041, A1043, A1073, A1393, and A1470) out of 17 selected strains are belonged to Streptomyces group, that possess specific and typical morphological characteristics that can be used as basic criteria for taxonomical identification [11], [29] A1073very different in shape (round or The colonies of these strains were ellipsoidal), surfaces roughness (smooth, ridged, wrinkled or granular), and profile (raised or flat) These colonies formed both substrate and aerial mycelia of various colors such as white, yellow, orange, red, and brown Colonies were completely covered with a leathery layer of aerial mycelia or concentric lines The size of colonies depended on the strains, age and varying from some millimeter up to approximately one centimeter (Fig 9) A1018 A390 A1073 A1043 Figure 9: Colony morphology of the representative Streptomycete strains 42 In regard to the formation of aerial mycelia or the formation of spore chains, 10 Streptomyces strains above produced aerial mycelia with long chains of spores on YS agar medium Characteristics of the spore bearing hyphae and spore chains were observed under phase contrast microscopy (Fig 10) (A) A1018 (B) A390 (C) A1073 (D) A1043 Figure 10: Spore-bearing aerial hyphae of the representative Streptomyces strains A, B - Rectiflexibiles type & C, D - Spirals type of spore bearing hyphae Based on microscopic observation, spore chain of these Streptomyces strains could be differentiated into two types: - Rectiflexibiles type (Fig 10A and B) contained strainght or flexuous spore chains, branched white aerial mycelia Related to this type were strains A390, A427, A1018, A1022, A1393 and A1470 - Spirals type (Fig 10 C and D) had aerial mycelia that formed branched spore-bearing hyphae with shape of hooks, open loops, open, loose, long, stretched spirals (Fig 10 C) or closed, compact spirals with 3-5 curves (Fig 10 D) Related to this type were strains A410, A1041, A1043 and A1073 43 The spores were oval to cyclindral in shape, arranged in long chains Special structures such as sporangia and sclerotia were not observed 3.5.2 16S rDNA sequence-based identification for the non-Streptomyces isolates Regarding the non-Streptomyces group, morphological characteristics are not allowed to distinguish the different strains like described above Sequencing and comparing of the 16S rDNA was used to identify the genetic relationship between selected strains The results obtained from phylogenetic analysis showed that all of them affiliated to the genus Nonomuraea and could make five different genetic groups within this genus (Fig 11) Figure 11: Neighbor-joining tree of 16S rDNA partial sequences showing phylogenetic positions of the actinomycete strains in the relationship to type strains of the genus Nonomuraea Bootstrap values of more than 500 are given at branching points Bar = 0.05 nucleotide divergence in the DNA sequences Microbispora parva was chosen as outgroup Thus, the actinomycete collection from Catba island had relatively high taxonomical diversity, however the selected strains with the highest 44 antimicrobial activity affiliated to only two genera Streptomyces and Nonomuraea Moreover, the three strains with cytotocicity against human cell lines belonged exclusively to the genus Streptomyces CONCLUSION AND SUGGESTION 1) The actinomycetes are well known as a producer of many useful secondary metabolites, including antibiotics The isolation of actinomycetes in nature is the first step of screening for natural products In this study, a total of 424 actinomycete strains were isolated from soil and litter samples on Catba island (Haiphong, Vietnam) by general dry–heating and specific rehydration–centrifugation methods 2) Screening of all the actinomycete isolates yielded high proportion of antibiotic producing strains (115 of 424, about 27%), among those 17 best strains were selected according to their significant inhibitory activity against the target microorganisms (including E coli, M luteus, F oxysporium and C albicans) TLC and HPLC analyses indicated that antibiotic substances produced by the selected strains were diverse in chemical nature, therefore could serve as good potential sources for searching new metabolites for drug development Additionally, the positive effects against human cell lines observed in three strains (A1018, A1022 and A1073) among the 17 selected strains would also be quite promising for discovering antitumor substances 3) Taxonomically, about 70% of the actinomycetes strains isolated from Catba island belonged to the Streptomyces group, the rest belonged to nonStreptomyces group which was dominated by Micromonospora, Nonomureae and Nocardia genera The 17 selected strains with the highest antimicrobial activity affiliated just to Streptomyces (10 strains) and Nonomurea genera (7 strains) Besides the results obtained, many questions remain to be answered and new questions arise from this work: 45 - Among the 115 isolates having significant inhibitory effect against the target microbial strains, only 17 strains were studied in details, the remaining strains are still of interest for study in order to discover new useful compounds - Crude extracts of three strains A1018, A1022 and A1073 exhibited significant cytotoxic activity against hepatocellular carcinoma, lung cancer, and rhabdosarcoma cell lines Further studies are required to investigate the antibiotics produced by these strains in term of activity spectra and chemical structures These could be potential sources of anticancer agents 46 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Yamazaki T., Iimura Y (2000), “Application of method incorporating differential centrifugation for selective isolation of motile actinomycetes in soil and plant litter”, Antonie Van Leeuwenhoek, 78, pp 171 – 185 16 Horie M., Saito K., Nakazama H (1996), “Determination of kitasamycin and josamycin in meat by HPLC”, Japan Analyst, 45, pp 1089 – 1094 17 Hopwood A D (2007), Streptomyces in nature and medicine, the antibiotics maker, Oxford University Press, pp 28 – 50 18 Hozzein W.N., Li W.J., Ali M.I.A., Hammouda O., Mousa A.S., Xu L.H., Jiang C.L (2004), “Nocardiopsis alkaliphila sp nov., a novel alkaliphilic actinomycete isolated from desert soil in Egypt”, Int J Syst Evol Microbiol, 54, pp 247-252 19 Ichikawa T., Date M., Ishikura T., Ozaki A (1971), “Improvement of kasugamycin-producing strain by the agar piece method and the prototroph method”, Folia Microbiol, 16, pp 218 – 224 20 Irena C (2010), Encyclopedia of chromatography 3rd Edition, CRC Press, pp 89 – 95 21 Jeffrey R.K (1978), 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NRRL 8167”, J Antibiot, 32, pp 100-107 46 Young S.R., Wan J.K., Dong J.Y., Heun S.K., Soon R.C (2001), “Synthesis and antitumor activity of new anthracycline analogues”, Bull Korean Chem Soc, Vol 22, 9, pp 963 – 968 51 APPENDIX I: Culture media Mueller – Hinton medium  (MHA): o Meat extraction  Yeast – Starch medium (YS): o Glucose 3g 10 g o Yeast extract 2g o Hydrolysis casein 17.5 g o Agar o Starch 1.5 g o Distilled water 1L o Agar 17 g o pH adjusted to 7.0 o Distilled water 17 g 1L o pH 7.4  Soybean meal medium: o Starch 25g o Soybean 15g o Yeast extract 1g o CaCO3 4g o Distilled water 1L o pH adjusted to 6.2 Yeast Malt medium  (YM): Malt extract o 3g Yeast extract o 3g Glucose o 10 g Pepton o 5g o Agar o Distilled water 52 17 g 1L Humic acid - vitamin agar  (HV agar) o Humid acid 1g o CaCO3 0.02 g o FeSO4.7H2O 0.01 g o KCl 1.71 g o MgSO4.7H2O 0.05 g o Na2HPO4 0.5 g o Cycloheximine 50 mg o Nalidixic acid 20 mg o Kabicidine 14 mg o Agar o pH 18 g 7.2 APPENDIX II: The 16S rDNA sequence >A0045 Contig1, 1495 bases TTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGGAAAGGCC CTTCGGGGTACTCGAGCGGCGAACGGGTGAGTAACACGTGAGCAACCTGCCCCTGACTCTGGGA TAAGCCCGGGAAACTGGGTCTAATACCGGATACGACCGCCCCCGGCATCGGGTGGTGGTGGAAA GTTTTTTCGGTTGGGGATGGGCTCGCGGCCTATCAGCTTGTTGGTGGGGTAGTGGCCTACCAAGG CGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGAC TCCTACGGGAGGCAGCAGTGGGGAATATTGCGCAATGGGCGGAAGCCTGACGCAGCGACGCCGC GTGGGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGACGAAGTTGACGTGTACCTG CAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTC CGGAATTATTGGGCGTAAAGAGCTCGTAGGTGGCTGGTCGCGTCTGCCGTGAAAGCCCGCAGCTT AACTGCGGGTCTGCGGTGGATACGGGCCGGCTAGAGGTAGGTAGGGGCAAGTGGAATTCCTGGT GTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGCTTGCTGGGCCTTA CCTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCTG TAAACGTTGGGCGCTAGGTGTGGGGATCTTCCACGATCTCCGTGCCGGAGCTAACGCATTAAGCG CCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAG CGGCGGAGCATGTTGCTTAATTCGACGCAACGCGAAGAACCTTACCAAGGTTTGACATCACCCG GAAACGCTCAGAGATGGGCGCCTCTTCGGACTGGGTGACAGGTGGTGCATGGCTGTCGTCAGCT CGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGCTCCATGTTGCCAGCACG CCCTTCGGGGTGGTGGGGACTCATGGGGGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATG ACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCAAACATGCTACAATGGCCGGTACAGAGGG TTGCGATACCGTGAGGTGGAGCGAATCCCTAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAA CTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAATGCTGCGGTGAATACGTTCC CGGGCCTTGTACACACCGCCCGTCACGTCACGAAAGTCGGCAACACCCGAAGCCCGTGGCCCAA CCACTTTGTGGGGGGAGCGGTCGAAGGTGGGGCTGGCGATTGGGACGAAGTCGTAACAAGGTA GCCGTACCGGAAGGTGCGGCT >A0149 Contig1, 1436 bases 53 TGCTTAACACATGCAAGTCGAGCGGAAAGGCCCTTCGGGGTACTCGAGCGGCGAACGGGTGAGT AACACGTGAGCAACCTGCCCCTGACTCTGGGATAAGCCCGGGAAACTGGGTCTAATACCGGATA CGACCGCCCCCGGCATCGGGTGGTGGTGGAAAGTTTTTTCGGTTGGGGATGGGCTCGCGGCCTAT CAGCTTGTTGGTGGGGTAGTGGCCTACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGAC CGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGC GCAATGGGCGGAAGCCTGACGCAGCGACGCCGCGTGGGGGATGACGGCCTTCGGGTTGTAAAC CTCTTTCAGCAGGGACGAAGTTGACGTGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAG CAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGT GGCTGGTCGCGTCTGCCGTGAAAGCCCGCAGCTTAACTGCGGGTCTGCGGTGGATACGGGCCGG CTAGAGGTAGGTAGGGGCAAGTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGG AACACCGGTGGCGAAGGCGGCTTGCTGGGCCTTACCTGACGCTGAGGAGCGAAAGCGTGGGGA GCGAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGTTGGGCGCTAGGTGTGGGGATCTT CCACGATCTCCGTGCCGGAGCTAACGCATTAAGCGCCCCGCCTGGGGAGTACGGCCGCAAGGCT AAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTTGCTTAATTCGACGCAA CGCGAAGAACCTTACCAAGGTTTGACATCACCCGGAAACGCCTGGAGACAGGCGCCTCTTCGGA CTGGGTGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCA ACGAGCGCAACCCTTGCTCCATGTTGCCAGCACGCCCTTCGGGGTGGTGGGGACTCATGGGGGA CTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGTCTTGGG CTGCAAACATGCTACAATGGCCGGTACAGAGGGTTGCGATACCGTGAGGTGGAGCGAATCCCTA AAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAAT CGCAGATCAGCAATGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCACG AAAGTCGGCAACACCCGAAGCCCGTGGCCCAACCACTTTGTGGGGGGAGCGGTCGAAGGTGGG GCTGGCGATTGGGACGAAGTCGTAAC >A0154 Contig1, 1476 bases CATGCAAGTCGAGCGGAAAGGCCCTTCGGGGTACTCGAGCGGCGAACGGGTGAGTAACACGTG AGCAACCTGCCCCTGACTCTGGGATAAGCCCGGGAAACTGGGTCTAATACCGGATACGACGTTCT GTCGCATGACATGAACGTGGAAAGTTTTTTCGGTCGGGGATGGGCTCGCGGCCTATCAGCTTGTT GGTGGGGTAGTGGCCTACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACAC TGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCGCAATGGGCG GAAGCCTGACGCAGCGACGCCGCGTGGGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCA GGGACGAAGTTGACGTGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGT AATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGTGGCTGGTCGC GTCTGCCGTGAAAGCCCGCAGCTTAACTGCGGGTCTGCGGTGGATACGGGCCGGCTAGAGGTAG GTAGGGGCAAGCGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTG GCGAAGGCGGCTTGCTGGGCCTTACCTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGA TTAGATACCCTGGTAGTCCACGCTGTAAACGTTGGGCGCTAGGTGTGGGACCCTTCCACGGGTTC CGTGCCGGAGCTAACGCATTAAGCGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAA GGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTTGCTTAATTCGACGCAACGCGAAGAAC CTTACCAAGGTTTGACATCACCCGGAAAGCTCTGGAGACAGAGCCCTCTTCGGACTGGGTGACA GGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCA ACCCTTGCTCCATGTTGCCAGCACGCCCTTCTACGTGGTGGGGACTCATGGGGGACTGCCGGGGT CAACTCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCAAACAT GCTACAATGGCCGGTACAGAGGGCTGCTAAGCCGTGAGGCGGAGCGAATCCCTAAAAGCCGGTC TCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAG CAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCACGAAAGTCGGC AACACCCGAAGCCCGTGGGCCAACCCCCAAGGGGGGGAGAGGGCGAAAGTGGGGCTGGCGATT GTGACAAAATCGTAACAAGGTAGCCGTACCGCAAAGTGCGGCTGGATCACCTCCTTATAAAGGA GC >A0160 Contig1, 1440 bases, TGCTTAACACATGCAAGTCGAGCGGAAAGGCCCTTCGGGGTACTCGAGCGGCGAACGGGTGAGT AACACGTGAGCAACCTGCCCCTGACTCTGGGATAAGCCCGGGAAACTGGGTCTAATACCGGATAT GACCGCCTCCGGCATCGGATGGTGGTGGAAAGTTTTTCGGTCGGGGATGGGCTCGCGGCCTATCA GCTTGTTGGTGGGGTAGTGGCCTACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCG GCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCGC AATGGGCGGAAGCCTGACGCAGCGACGCCGCGTGGGGGATGACGGCCTTCGGGTTGTAAACCTC TTTCAGCAGGGACGAAGTTGACGTGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAG CCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGTGGC TGGTCGCGTCTGCCGTGAAAGCCCGCAGCTTAACTGCGGGTCTGCGGTGGATACGGGCCGGCTA GAGGTAGGTAGGGGCAAGTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAAC ACCGGTGGCGAAGGCGGCTTGCTGGGCCTTACCTGACGCTGAGGAGCGAAAGCGTGGGGAGCG 54 AACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGTTGGGCGCTAGGTGTGGGACCCTTCCA CGGGTTCCGTGCCGGAGCTAACGCATTAAGCGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAA ACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTTGCTTAATTCGACGCAACGC GAAGAACCTTACCAAGGTTTGACATCACCCGGACCGGTCCAGAGATAGGCCTTCCCTTTTGGGCT GGGTGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAA CGAGCGCAACCCTTGCTCCATGTTGCCAGCAACACCTTCGGGTGGTTGGGGACTCATGGGGGAC TGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGTCTTGGGC TGCAAACATGCTACAATGGCCGGTACAGAGGGTTGCGATACCGTGAGGTGGAGCGAATCCCTAA AAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATC GCAGATCAGCAATGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCACGA AAGTCGGCAACACCCGAAGCCCGTGGCCCAACCAGCTTGCTGGGGGGAGCGGTCGAAGGTGGG GCTGGCGATTGGGACGAAGTCGTAACAA >A0232 Contig1, 1441 bases GCGTGCTTAACACATGCAAGTCGAGCGGAAAGGCCCTTCGGGGTACTCGAGCGGCGAACGGGT GAGTAACACGTGAGCAACCTGCCCCTGACTCTGGGATAAGCCCGGGAAACTGGGTCTAATACCG GATACGACACACCCCGGCATCGGGTGTGTGTGGAAAGTTTTTCGGTTGGGGATGGGCTCGCGGC CTATCAGCTTGTTGGTGGGGTAGTGGCCTACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGC GACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATAT TGCGCAATGGGCGGAAGCCTGACGCAGCGACGCCGCGTGGGGGATGACGGCCTTCGGGTTGTAA ACCTCTTTCAGCAGGGACGAAGTTGACGTGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCC AGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTA GGTGGCTGGTCGCGTCTGCCGTGAAAGCCCGCAGCTTAACTGCGGGTCTGCGGTGGATACGGGC CGGCTAGAGGTAGGCAGGGGCAAGTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGG AGGAACACCGGTGGCGAAGGCGGCTTGCTGGGCCTTACCTGACGCTGAGGAGCGAAAGCGTGG GGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGTTGGGCGCTAGGTGTGGGAC CCTTCCACGGGTTCCGTGCCGGAGCTAACGCATTAAGCGCCCCGCCTGGGGAGTACGGCCGCAA GGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTTGCTTAATTCGAC GCAACGCGAAGAACCTTACCAAGGTTTGACATCACCCGGAAACGGCCAGAGATGGTCGCCTCTT CGGACTGGGTGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTC CCGCAACGAGCGCAACCCTTGTTCCATGTTGCCAGCACGCCCTTCGGGGTGGTGGGGACTCATG GGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGT CTTGGGCTGCAAACATGCTACAATGGCCGGTACAGAGGGCTGCTAAACCGCGAGGTGGAGCGAA TCCCTAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCT AGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCAC GTCACGAAAGTCGGCAACACCCGAAGCCCGTGGCCCAACCAGCTTGCTGGGGGGAGCGGTCGA AGGTGGGGCTGGCGATTGGGACGAAGTCGTAACAA >A0396 Contig1, 1432 bases TGCTTAACACATGCAAGTCGAGCGGAAAGGCCCTTCGGGGTACTCGAGCGGCGAACGGGTGAGT AACACGTGAGCAACCTGCCCCTGACTCTGGGATAAGCCCGGGAAACTGGGTCTAATACCGGATAT GACCGCCTCCGGCATCGGATGGTGGTGGAAAGTTTTTCGGTCGGGGATGGGCTCGCGGCCTATCA GCTTGTTGGTGGGGTAGTGGCCTACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCG GCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCGC AATGGGCGGAAGCCTGACGCAGCGACGCCGCGTGGGGGATGACGGCCTTCGGGTTGTAAACCTC TTTCAGCAGGGACGAAGTTGACGTGTACCTGCAGAAGAAGCGCCGGCTAACTACGTGCCAGCAG CCGCGGTAATACGTAGGGCGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGAGCTCGTAGGTGGC TGGTCGCGTCTGCCGTGAAAGCCCGCAGCTTAACTGCGGGTCTGCGGTGGATACGGGCCGGCTA GAGGTAGGTAGGGGCAAGTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAAC ACCGGTGGCGAAGGCGGCTTGCTGGGCCTTACCTGACGCTGAGGAGCGAAAGCGTGGGGAGCG AACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGTTGGGCGCTAGGTGTGGGACCCTTCCA CGGGTTCCGTGCCGGAGCTAACGCATTAAGCGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAA ACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTTGCTTAATTCGACGCAACGC GAAGAACCTTACCAAGGTTTGACATCACCCGGACCGCCTCAGAGATGGGGCTTCCCTTTTGGGCT GGGTGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAA CGAGCGCAACCCTTGCTCCATGTTGCCAGCAACACCTTCGGGTGGTTGGGGACTCATGGGGGAC TGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGTCTTGGGC TGCAAACATGCTACAATGGCCGGTACAGAGGGTTGCGATACCGTGAGGTGGAGCGAATCCCTAA AAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATC GCAGATCAGCAATGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCACGA AAGTCGGCAACACCCGAAGCCCGTGGCCCAACCAGCTTGCTGGGGGGAGCGGTCGAAGGTGGG GCTGGCGATTGGGACGAAGT 55 >A0444 Contig1, 1496 bases GTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAGCGGAAAGG CCCTTCGGGGTACTCGAGCGGCGAACGGGTGAGTAACACGTGAGCAACCTGCCCCTGACTCTGG GATAAGCCCGGGAAACTGGGTCTAATACCGGATATGACCGCCTCCGGCATCGGATGGTGGTGGAA AGTTTTTCGGTTGGGGATGGGCTCGCGGCCTATCAGCTTGTTGGTGGGGTAGTGGCCTACCAAGG CGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGAC TCCTACGGGAGGCAGCAGTGGGGAATATTGCGCAATGGGCGGAAGCCTGACGCAGCGACGCCGC GTGGGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCAGGGACGAAGTTGACGTGTACCTG CAGAAGAAGCGCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGCGCAAGCGTTGTC CGGAATTATTGGGCGTAAAGAGCTCGTAGGTGGCTGGTCGCGTCTGCCGTGAAAGCCCGCAGCTT AACTGCGGGTCTGCGGTGGATACGGGCCGGCTAGAGGTAGGTAGGGGCAAGTGGAATTCCTGGT GTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGCTTGCTGGGCCTTA CCTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCTG TAAACGTTGGGCGCTAGGTGTGGGACCCTTCCACGGGTTCCGTGCCGGAGCTAACGCATTAAGC GCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAA GCGGCGGAGCATGTTGCTTAATTCGACGCAACGCGAAGAACCTTACCAAGGTTTGACATCACCC GGACCGGTCTAGAGATAGGCCTTCCCTTTTGGGCTGGGTGACAGGTGGTGCATGGCTGTCGTCAG CTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGCTCCATGTTGCCAGCA ACACTTTCGGGTGGTTGGGGACTCATGGGGGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGA TGACGTCAAGTCATCATGCCCCTTATGTCTTGGGCTGCAAACATGCTACAATGGCCGGTACAGAG GGTTGCGATACCGTGAGGTGGAGCGAATCCCTAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGC AACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAATGCTGCGGTGAATACGTT CCCGGGCCTTGTACACACCGCCCGTCACGTCACGAAAGTCGGCAACACCCGAAGCCCGTGGCCC AACCAGCTTGCTGGGGGGAGCGGTCGAAGGTGGGGCTGGCGATTGGGACGAAGTCGTAACAAG GTAGCCGTACCGGAAGGTGCG 56 ... indicated that the collection of actinomycetes isolated from Catba island contained mainly Streptomyces species (about 70%) and the group of rare actinomycetes (non-Streptomyces) which made of. .. been investigated on microbial diversity and their potential use The study “Biodiversity and antibiotic activity of actinomycetes isolated from Catba island, Vietnam? ?? presented here was conducted... flow Collection and isolation of actinomycetes Collection of samples from Catba island Isolation by dry – heating and rehydration – centrifugation Screening of antibiotic producing actinomycetes