Screening for Antibiotics from Indigenous Streptomycetes, Their Genetic and Mutational Analysis

Total Page:16

File Type:pdf, Size:1020Kb

Screening for Antibiotics from Indigenous Streptomycetes, Their Genetic and Mutational Analysis SCREENING FOR ANTIBIOTICS FROM INDIGENOUS STREPTOMYCETES, THEIR GENETIC AND MUTATIONAL ANALYSIS By IMRAN SAJID April 2009 DEPARTMENT OF MICROBIOLOGY AND MOLECULAR GENETICS A THESIS SUBMITTED TO THE UNIVERSITY OF THE PUNJAB, IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEDICATION This work is dedicated to my late grandfather “Chaudhary Muhammad Ismail”. i ACKNOWLEDGMENTS All the praises and thanks are for the most merciful and most compassionate, Al-mighty Allah, who is the entire source of all knowledge and wisdom. It is He, who blessed me the courage, ability, patience and sufficient opportunity to complete this work. It is my privilege and honour to express my deepest gratitude to my distinguished teacher and research supervisor Prof. Dr. Shahida Hasnain, Chairperson, Department of Microbiology and Molecular Genetics, Dean Faculty of Life Sciences, University of the Punjab, Quaid-e-Azam, Campus, Lahore, for her inspiring guidance, useful and intellectual suggestions, timely advices and encouragement. Her keen interest and painstaking efforts during the course of research work are specially acknowledged. My special thanks go to Prof. Dr. Hartmut Laatsch, Institute of Organic and Biomolecular Chemistry, University of Göttingen, Germany, for his cooperation and guidance during my stay in his research group. I am also thankful to Prof. Dr Hans Joachim Fritz and Dr Bloga Popava, Department of Molecular Genetics and Preparative Molecular Biology, University of Göttingen, Germany, Dr Hans Joachim Shindler (Germany), Dr Anjum Naseem Sabri, Dr Muhammad Faisal and Dr Sikandar Sulatn, Department of Microbiology and Molecular Genetics, University of the Punjab, Lahore, Pakistan, for their sincere help providing facilities, cooperation, moral support and valuable discussions. I feel happy to thank my research colleagues and friends, Dr Kahaled A Shabaan, Dr Hafiz-ur-Rahman, Dr Claresse Blandine Fotso Fondga Yao, and MS Zindha Imene in Germany, Mr Basharat Ali, Dr Farrukh M Alvi, Mr Anwar Hussain, Mr Mahboob Ahmed, Ms Rida Batool, Ms Ambreen Ahmed, Mr Kashif, Dr Najma shaheen, Dr Fouzia Qamar and Mrs Farkhanda Jabeen, here in Pakistan, for their charming company and support during my research work. I am also grateful to my family members for their prayers, love and moral support at every step of my life. The financial support for this work provided by Higher Education Commission of Pakistan (HEC) under IRSIP is gratefully acknowledged. Imran Sajid ii CONTENTS S.NO. Description Page No. Dedication i Acknowledgements ii Contents iii List of Abbreviations iv List of Tables v List of Figures vii Summary xi Chapter 1 Introduction 1 Chapter 2 Materials and Methods 20 Chapter 3 Isolation and characterization of indigenous 64 bioactive streptomycetes Chapter 4 Prescreening (Biological and Chemical screening) 85 Chapter 5 Fermentation, isolation, purification and structure 116 elucidation of the metabolites Chapter 6 16S rRNA gene sequence analysis 153 Chapter 7 Culture Optimizations 168 Chapter 8 Mutational Analysis 180 Chapter 9 Discussion 210 Chapter 10 References 224 Supplemental Data 238 Publications 251 iii LIST OF ABBREVIATIONS AIA Actinomycetes isolation agar BLAST Basic local alignment and search tool bp Base pair(s) CFU Colony Forming Unit Cm Chloramphenicol DDW Double distilled water DCM Dichloromethane DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid EB Ethidium Bromide EDTA Ethylenediamine-N,N,N',N'-tetraacetic acid EI-MS Electron impact mass spectrometry EMS ethane methane sulphonates ESI-MS Electro spray ionization mass spectrometry GYM Glucose, Yeast & Malt extract medium HPLC-MS High performance liquid chromatography mass spectrometry HR-MS High resolution mass spectrometry J Coupling constant m/z Mass-to-charge ratio MS Mass Spectrometry MeOH Methanol MNNG N-methyl-N'-nitro-N-nitrosoguanidine NCBI National centre for biotechnology information NMR Nuclear magnetic resonance PCR Polymerase chain reaction Rf Relative front Rt Retention time RPM Revolution per minutes SPE Solid phase extraction TLC Thin layer chromatography UV Ultra violet δ Chemical shift iv LIST OF TABLES S. No. Title Page No. Table 3.1 Soil samples collected for the isolation of indigenous 67 streptomycetes Table 3.2 Group 1: Streptomyces strains isolated from the soil of 70 rose field and botanical garden and selected on the basis of preliminary antimicrobial activity Table 3.3 Group 2: Streptomyces strains isolated from the soil of 71 corn field (saline soil) and selected on the basis of preliminary antimicrobial activity Table 3.4 Group 3: Streptomyces strains isolated from the soil of 72 cotton field (saline soil) and selected on the basis of preliminary antimicrobial activity Table 3.5 Group 4: Streptomyces strains isolated from the soil of a 73 sugar cane field (saline soil) and selected on the basis of preliminary antimicrobial activity Table 3.6 Group 5: Streptomyces strains isolated from the soil of 74 northern areas of Pakistan (forest soil) and selected on the basis of preliminary antimicrobial activity Table: 3.7 Morphological characteristics of the selected 75 Streptomyces strains after incubation at 28˚C for 7 days on GYM agar plates Table: 3.8 Growth temperature range of the selected Streptomyces 79 strains Table: 3.9 Melanin production and sugar utilization profile of the 80 selected strains Table: 3.10 Results of utilization of organic acids, oxalates, 81 hydrolysis of urea and hemolysis test of the selected Streptomyces strains Table 4.1 Results of screening of Streptomyces strains for 88 antimicrobial activity and cytotoxicity Table 4.2 Results of HPLC-MS/MS analysis 92 Table 5.1 Physico-chemical properties of geninthiocin (1) and val- 119 geninthiocin (2) Table 5.2 13C and 1H NMR of geninthiocin (1) and val- 120 geninthiocin (2) compared with literature in DMSO-d6 Table 5.3 MS2 and MS3 product ions of the [M+H]+ precursor ions 126 of geninthiocin (1) and val-geninthiocin (2) obtained on a quadrupol ion trap mass spectrometer Table 5.4 Confirmation of key fragments by exact mass 127 determination using a FT-ICR mass spectrometer Table 5.5 Antibacterial and antifungal activities of val- 127 geninthiocin (1) in comparison with geninthiocin (2) and v chalcomycin (conc. in 40µg/ disk) Table 6.1 Gene bank accession numbers along with the alignments 155 of sequences obtained with reported 16S rRNA gene sequences in the gene bank and highest similarity with different Streptomyces species (17 strains) Table 7.1 Comparison of the Wt. of crude extracts/250 ml culture 171 broth of the strains RSF 18 and CTF9 for six different media compositions and four different sets of culture conditions Table 7.2 Comparison of the antimicrobial activity of the strains 174 RSF18 and CTF9 at six different media compositions and four different sets of culture conditions Table 8.1 Antibiotic sensitivity pattern of wild type strain 182 Streptomyces sp. RSF18 against various antibiotics Table 8.2 MTC of Chloramphenicol for wild type strain 182 Streptomyces sp. RSF18 Table 8.3 Number of chloramphenicol resistant mutant colonies 185 obtained by ethidium bromide treatments Table 8.4 Antimicrobial activity of the chloramphenicol resistant 185 mutants obtained by ethidium bromide treatments in comparison with wild strain against Bacillus subtilus Table 8.5 Number of chloramphenicol resistant mutants colonies 187 from different MNNG treatments Table 8.6 Antimicrobial activity of the chloramphenicol resistant 187 mutants obtained by MNNG treatments in comparison with wild strain against Bacillus subtilus Table 8.7 Number of Chloramphenicol resistant mutant colonies 191 obtained by UV irradiation Table 8.8 Antimicrobial activity of chloramphenicol resistant 191 mutants obtained by UV irradiation in comparison with wild strain against Bacillus subtilus Table 8.9 Antimicrobial activity at different time intervals of 194 randomly selected derivatives of RSF18 after treatment with gamma radiations at different intensities vi LIST OF FIGURES S. No Title Page No Figure 3.1 Working up scheme for the isolation and characterization 66 of indigenous Streptomyces strains from soil Figure 3.2 Crowding of Streptomyces on actinomycetes isolation agar 71 medium (AIA) Figure 3.3 Preliminary antimicrobial activities of the Streptomyces 74 isolates determined by the solid media bioassay Figure 3.4 Some selected Streptomyces strains exhibiting variations 76 in morphological characteristics. Figure 3.5 Microscopic images of some selected Streptomyces strains 77 Figure 4.1 Working up scheme for the pre-screening of selected 87 Streptomyces strains Figure 4.2 Antimicrobial activities of selected Streptomyces strains 90 against different test organisms Figure 4.3 Chemical screening using TLC with anisaldehyde/H2SO4 91 and Ehrlich’s spraying reagents Figure 4.4a Total ion chromatogram of crude extract RSF17 96 Figure 4.4b Selected ion chromatogram of extract RSF17 96 Figure 4.5a Total ion chromatogram of crude extract RSF18 97 Figure 4.5b Selected ion chromatogram of extract RSF18 97 Figure 4.6a Total ion chromatogram of crude extract RSF23 98 Figure 4.6b Selected ion chromatogram of extract RSF23 98 Figure 4.7a Total ion chromatogram of crude extract BG5 99 Figure 4.7b Selected ion chromatogram of extract BG5 99 Figure 4.8a Total ion chromatogram of crude extract CRF 100 Figure 4.8b Selected ion chromatogram of extract CRF1 100 Figure 4.9a Total ion chromatogram of crude extract CRF2 101 Figure 4.9b Selected ion chromatogram of extract CRF2 101 Figure 4.10a Total
Recommended publications
  • Identification and Antibiosis of a Novel Actinomycete Strain RAF-11 Isolated from Iraqi Soil
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by GSSRR.ORG: International Journals: Publishing Research Papers in all Fields International Journal of Sciences: Basic and Applied Research (IJSBAR) ISSN 2307-4531 http://gssrr.org/index.php?journal=JournalOfBasicAndApplied Identification and Antibiosis of a Novel Actinomycete Strain RAF-11 Isolated From Iraqi Soil. R. FORAR. LAIDIa, A. ABDERRAHMANEb, A. A. HOCINE NORYAc. a Department of Natural Sciences, Ecole Normale Superieure, Vieux-Kouba, Algiers – Algeria b,c Institute of Genetic Engineering and Biotechnology, Baghdad - Iraq. a email: [email protected] Abstract A total of 35 actinomycetes strains were isolated from and around Baghdad, Iraq, at a depth of 5-10 m, by serial dilution agar plating method. Nineteen out of them showed noticeable antimicrobial activities against at least, to one of the target pathogens. Five among the nineteen were active against both Gram positive and Gram negative bacteria, yeasts and moulds. The most active isolate, strain RAF-11, based on its largest zone of inhibition and strong antifungal activity, especially against Candida albicans and Aspergillus niger, the causative of candidiasis and aspergillosis respectively, was selected for identification. Morphological and chemical studies indicated that this isolate belongs to the genus Streptomyces. Analysis of the 16S rDNA sequence showed a high similarity, 98 %, with the most closely related species, Streptomyces labedae NBRC 15864T/AB184704, S. erythrogriseus LMG 19406T/AJ781328, S. griseoincarnatus LMG 19316T/AJ781321 and S. variabilis NBRC 12825T/AB184884, having the closest match. From the taxonomic features, strain RAF-11 matched with S. labedae, in the morphological, physiological and biochemical characters, however it showed significant differences in morphological characteristics with this nearest species, S.
    [Show full text]
  • A Bioactive Fraction from Streptomyces Sp. Enhances Maize Tolerance Against Drought Stress
    J. Microbiol. Biotechnol. 2020. 30(8): 1156–1168 https://doi.org/10.4014/jmb.2003.03034 A Bioactive Fraction from Streptomyces sp. Enhances Maize Tolerance against Drought Stress Mona Warrad1*, Yasser M. Hassan2*, Mahmoud S.M. Mohamed3, Nashwa Hagagy4,7, Omar A. Al-Maghrabi5, Samy Selim6,7, Ahmed M. Saleh8, and Hamada AbdElgawad2 1Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Al-Qurayyat, 2014, Jouf University, Saudi Arabia 2Department of Botany and Microbiology, Faculty of Science, Beni-Suef University, Beni-Suef 62521, Egypt 3Department of Botany and Microbiology, Faculty of Science, Cairo University, Giza 12613, Egypt 4Department of Biology, College of Science and Arts at Khulis, University of Jeddah, Jeddah 21959, Saudi Arabia 5Department of Biology, College of Science, University of Jeddah, Jeddah 21959, Saudi Arabia 6Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Jouf University, Sakaka, P.O. 2014, Saudi Arabia 7Botany Department, Faculty of Science, Suez Canal University, Ismailia 41522, Egypt 8Biology Department, Faculty of Science at Yanbu, Taibah University, King Khalid Rd., Al Amoedi, 46423, Yanbu El- Bahr, Saudi Arabia Drought stress is threatening the growth and productivity of many economical crops. Therefore, it is necessary to establish innovative and efficient approaches for improving crop growth and productivity. Here we investigated the potentials of the cell-free extract of Actinobacteria (Ac) isolated from a semi-arid habitat (Al-Jouf region, Saudi Arabia) to recover the reduction in maize growth and improve the physiological stress tolerance induced by drought. Three Ac isolates were screened for production of secondary metabolites, antioxidant and antimicrobial activities. The isolate Ac3 revealed the highest levels of flavonoids, antioxidant and antimicrobial activities in addition to having abilities to produce siderophores and phytohormones.
    [Show full text]
  • Isolation and Identification of Streptomyces Rochei Strain Active Against Phytopathogenic Fungi
    British Microbiology Research Journal 4(10): 1057-1068, 2014 SCIENCEDOMAIN international www.sciencedomain.org Isolation and Identification of Streptomyces rochei Strain Active against Phytopathogenic Fungi Adil A. El Hussein1*, Rihab E. M. Alhasan2, Suhair A. Abdelwahab3 and Marmar A. El Siddig1 1Department of Botany, Faculty of Science, University of Khartoum, Sudan. 2Environmental and Natural Resources Research Institute, National Center for Research, Sudan. 3Biology Department, Duba University College, Tabuk University, Saudi Arabia. Authors’ contributions This work was carried out in collaboration between all authors. Author AAEH designed the study, performed the statistical analysis, wrote the protocol and prepared the final draft of the manuscript. Author REMA conducted the major parts of the practical work. Author SAA did the identification of the fungal species. Author MAES managed the literature searches, wrote the first draft of the manuscript and performed DNA analysis. All authors read and approved the final manuscript. Received 25th April 2014 th Original Research Article Accepted 18 May 2014 Published 7th June 2014 ABSTRACT A total of 104 actinomycete isolates were recovered from farming soil samples collected from 11 states in Sudan. Upon screening for potential antifungal activity, an actinomycete isolate (R92) was found to be highly antagonistic against all of the tested phytopathogenic fungi. It was identified as Streptomyces rochei on the basis of its morphology, chemotaxonomy and 16S rDNA sequence analysis. In vivo antagonistic activities of the n- butanol extract of R92 culture were significant since the progress of Drechslera halodes leaf spot on sorghum and Alternaria alternata early blight on tomato was highly restricted and incidence of both diseases was greatly suppressed.
    [Show full text]
  • Streptomyces As a Prominent Resource of Future Anti-MRSA Drugs
    REVIEW published: 24 September 2018 doi: 10.3389/fmicb.2018.02221 Streptomyces as a Prominent Resource of Future Anti-MRSA Drugs Hefa Mangzira Kemung 1,2, Loh Teng-Hern Tan 1,2,3, Tahir Mehmood Khan 1,2,4, Kok-Gan Chan 5,6*, Priyia Pusparajah 3*, Bey-Hing Goh 1,2,7* and Learn-Han Lee 1,2,3,7* 1 Novel Bacteria and Drug Discovery Research Group, Biomedicine Research Advancement Centre, School of Pharmacy, Monash University Malaysia, Bandar Sunway, Malaysia, 2 Biofunctional Molecule Exploratory Research Group, Biomedicine Research Advancement Centre, School of Pharmacy, Monash University Malaysia, Bandar Sunway, Malaysia, 3 Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Bandar Sunway, Malaysia, 4 The Institute of Pharmaceutical Sciences (IPS), University of Veterinary and Animal Sciences (UVAS), Lahore, Pakistan, 5 Division of Genetics and Molecular Biology, Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia, 6 International Genome Centre, Jiangsu University, Zhenjiang, China, 7 Center of Health Outcomes Research and Therapeutic Safety (Cohorts), School of Pharmaceutical Sciences, University of Phayao, Mueang Phayao, Thailand Methicillin-resistant Staphylococcus aureus (MRSA) pose a significant health threat as Edited by: they tend to cause severe infections in vulnerable populations and are difficult to treat Miklos Fuzi, due to a limited range of effective antibiotics and also their ability to form biofilm. These Semmelweis University, Hungary organisms were once limited to hospital acquired infections but are now widely present Reviewed by: Dipesh Dhakal, in the community and even in animals. Furthermore, these organisms are constantly Sun Moon University, South Korea evolving to develop resistance to more antibiotics.
    [Show full text]
  • This Article Appeared in a Journal Published by Elsevier. the Attached
    This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy ARTICLE IN PRESS Systematic and Applied Microbiology 32 (2009) 314–322 www.elsevier.de/syapm Proposal to reclassify the Streptomyces albidoflavus clade on the basis of multilocus sequence analysis and DNA–DNA hybridization, and taxonomic elucidation of Streptomyces griseus subsp. solvifaciens Xiaoying Rong, Yinping Guo, Ying Huangà State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China Received 22 April 2009 Abstract The Streptomyces albidoflavus 16S rRNA gene clade contains 10 species and subspecies with identical 16S rRNA gene sequences and very similar numerical taxonomic data, including Streptomyces griseus subsp. solvifaciens. Type strains of this clade, as well as three CGMCC strains which were received as Streptomyces galilaeus, Streptomyces sioyaensis
    [Show full text]
  • Improved Taxonomy of the Genus Streptomyces
    UNIVERSITEIT GENT Faculteit Wetenschappen Vakgroep Biochemie, Fysiologie & Microbiologie Laboratorium voor Microbiologie Improved taxonomy of the genus Streptomyces Benjamin LANOOT Scriptie voorgelegd tot het behalen van de graad van Doctor in de Wetenschappen (Biochemie) Promotor: Prof. Dr. ir. J. Swings Co-promotor: Dr. M. Vancanneyt Academiejaar 2004-2005 FACULTY OF SCIENCES ____________________________________________________________ DEPARTMENT OF BIOCHEMISTRY, PHYSIOLOGY AND MICROBIOLOGY UNIVERSITEIT LABORATORY OF MICROBIOLOGY GENT IMPROVED TAXONOMY OF THE GENUS STREPTOMYCES DISSERTATION Submitted in fulfilment of the requirements for the degree of Doctor (Ph D) in Sciences, Biochemistry December 2004 Benjamin LANOOT Promotor: Prof. Dr. ir. J. SWINGS Co-promotor: Dr. M. VANCANNEYT 1: Aerial mycelium of a Streptomyces sp. © Michel Cavatta, Academy de Lyon, France 1 2 2: Streptomyces coelicolor colonies © John Innes Centre 3: Blue haloes surrounding Streptomyces coelicolor colonies are secreted 3 4 actinorhodin (an antibiotic) © John Innes Centre 4: Antibiotic droplet secreted by Streptomyces coelicolor © John Innes Centre PhD thesis, Faculty of Sciences, Ghent University, Ghent, Belgium. Publicly defended in Ghent, December 9th, 2004. Examination Commission PROF. DR. J. VAN BEEUMEN (ACTING CHAIRMAN) Faculty of Sciences, University of Ghent PROF. DR. IR. J. SWINGS (PROMOTOR) Faculty of Sciences, University of Ghent DR. M. VANCANNEYT (CO-PROMOTOR) Faculty of Sciences, University of Ghent PROF. DR. M. GOODFELLOW Department of Agricultural & Environmental Science University of Newcastle, UK PROF. Z. LIU Institute of Microbiology Chinese Academy of Sciences, Beijing, P.R. China DR. D. LABEDA United States Department of Agriculture National Center for Agricultural Utilization Research Peoria, IL, USA PROF. DR. R.M. KROPPENSTEDT Deutsche Sammlung von Mikroorganismen & Zellkulturen (DSMZ) Braunschweig, Germany DR.
    [Show full text]
  • Study of Actinobacteria and Their Secondary Metabolites from Various Habitats in Indonesia and Deep-Sea of the North Atlantic Ocean
    Study of Actinobacteria and their Secondary Metabolites from Various Habitats in Indonesia and Deep-Sea of the North Atlantic Ocean Von der Fakultät für Lebenswissenschaften der Technischen Universität Carolo-Wilhelmina zu Braunschweig zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigte D i s s e r t a t i o n von Chandra Risdian aus Jakarta / Indonesien 1. Referent: Professor Dr. Michael Steinert 2. Referent: Privatdozent Dr. Joachim M. Wink eingereicht am: 18.12.2019 mündliche Prüfung (Disputation) am: 04.03.2020 Druckjahr 2020 ii Vorveröffentlichungen der Dissertation Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Fakultät für Lebenswissenschaften, vertreten durch den Mentor der Arbeit, in folgenden Beiträgen vorab veröffentlicht: Publikationen Risdian C, Primahana G, Mozef T, Dewi RT, Ratnakomala S, Lisdiyanti P, and Wink J. Screening of antimicrobial producing Actinobacteria from Enggano Island, Indonesia. AIP Conf Proc 2024(1):020039 (2018). Risdian C, Mozef T, and Wink J. Biosynthesis of polyketides in Streptomyces. Microorganisms 7(5):124 (2019) Posterbeiträge Risdian C, Mozef T, Dewi RT, Primahana G, Lisdiyanti P, Ratnakomala S, Sudarman E, Steinert M, and Wink J. Isolation, characterization, and screening of antibiotic producing Streptomyces spp. collected from soil of Enggano Island, Indonesia. The 7th HIPS Symposium, Saarbrücken, Germany (2017). Risdian C, Ratnakomala S, Lisdiyanti P, Mozef T, and Wink J. Multilocus sequence analysis of Streptomyces sp. SHP 1-2 and related species for phylogenetic and taxonomic studies. The HIPS Symposium, Saarbrücken, Germany (2019). iii Acknowledgements Acknowledgements First and foremost I would like to express my deep gratitude to my mentor PD Dr.
    [Show full text]
  • Alloactinosynnema Sp
    University of New Mexico UNM Digital Repository Chemistry ETDs Electronic Theses and Dissertations Summer 7-11-2017 AN INTEGRATED BIOINFORMATIC/ EXPERIMENTAL APPROACH FOR DISCOVERING NOVEL TYPE II POLYKETIDES ENCODED IN ACTINOBACTERIAL GENOMES Wubin Gao University of New Mexico Follow this and additional works at: https://digitalrepository.unm.edu/chem_etds Part of the Bioinformatics Commons, Chemistry Commons, and the Other Microbiology Commons Recommended Citation Gao, Wubin. "AN INTEGRATED BIOINFORMATIC/EXPERIMENTAL APPROACH FOR DISCOVERING NOVEL TYPE II POLYKETIDES ENCODED IN ACTINOBACTERIAL GENOMES." (2017). https://digitalrepository.unm.edu/chem_etds/73 This Dissertation is brought to you for free and open access by the Electronic Theses and Dissertations at UNM Digital Repository. It has been accepted for inclusion in Chemistry ETDs by an authorized administrator of UNM Digital Repository. For more information, please contact [email protected]. Wubin Gao Candidate Chemistry and Chemical Biology Department This dissertation is approved, and it is acceptable in quality and form for publication: Approved by the Dissertation Committee: Jeremy S. Edwards, Chairperson Charles E. Melançon III, Advisor Lina Cui Changjian (Jim) Feng i AN INTEGRATED BIOINFORMATIC/EXPERIMENTAL APPROACH FOR DISCOVERING NOVEL TYPE II POLYKETIDES ENCODED IN ACTINOBACTERIAL GENOMES by WUBIN GAO B.S., Bioengineering, China University of Mining and Technology, Beijing, 2012 DISSERTATION Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Chemistry The University of New Mexico Albuquerque, New Mexico July 2017 ii DEDICATION This dissertation is dedicated to my altruistic parents, Wannian Gao and Saifeng Li, who never stopped encouraging me to learn more and always supported my decisions on study and life.
    [Show full text]
  • Genomic and Phylogenomic Insights Into the Family Streptomycetaceae Lead to Proposal of Charcoactinosporaceae Fam. Nov. and 8 No
    bioRxiv preprint doi: https://doi.org/10.1101/2020.07.08.193797; this version posted July 8, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Genomic and phylogenomic insights into the family Streptomycetaceae 2 lead to proposal of Charcoactinosporaceae fam. nov. and 8 novel genera 3 with emended descriptions of Streptomyces calvus 4 Munusamy Madhaiyan1, †, * Venkatakrishnan Sivaraj Saravanan2, † Wah-Seng See-Too3, † 5 1Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, 6 Singapore 117604; 2Department of Microbiology, Indira Gandhi College of Arts and Science, 7 Kathirkamam 605009, Pondicherry, India; 3Division of Genetics and Molecular Biology, 8 Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, 9 Malaysia 10 *Corresponding author: Temasek Life Sciences Laboratory, 1 Research Link, National 11 University of Singapore, Singapore 117604; E-mail: [email protected] 12 †All these authors have contributed equally to this work 13 Abstract 14 Streptomycetaceae is one of the oldest families within phylum Actinobacteria and it is large and 15 diverse in terms of number of described taxa. The members of the family are known for their 16 ability to produce medically important secondary metabolites and antibiotics. In this study, 17 strains showing low 16S rRNA gene similarity (<97.3 %) with other members of 18 Streptomycetaceae were identified and subjected to phylogenomic analysis using 33 orthologous 19 gene clusters (OGC) for accurate taxonomic reassignment resulted in identification of eight 20 distinct and deeply branching clades, further average amino acid identity (AAI) analysis showed 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.08.193797; this version posted July 8, 2020.
    [Show full text]
  • Induction of Secondary Metabolism Across Actinobacterial Genera
    Induction of secondary metabolism across actinobacterial genera A thesis submitted for the award Doctor of Philosophy at Flinders University of South Australia Rio Risandiansyah Department of Medical Biotechnology Faculty of Medicine, Nursing and Health Sciences Flinders University 2016 TABLE OF CONTENTS TABLE OF CONTENTS ............................................................................................ ii TABLE OF FIGURES ............................................................................................. viii LIST OF TABLES .................................................................................................... xii SUMMARY ......................................................................................................... xiii DECLARATION ...................................................................................................... xv ACKNOWLEDGEMENTS ...................................................................................... xvi Chapter 1. Literature review ................................................................................. 1 1.1 Actinobacteria as a source of novel bioactive compounds ......................... 1 1.1.1 Natural product discovery from actinobacteria .................................... 1 1.1.2 The need for new antibiotics ............................................................... 3 1.1.3 Secondary metabolite biosynthetic pathways in actinobacteria ........... 4 1.1.4 Streptomyces genetic potential: cryptic/silent genes ..........................
    [Show full text]
  • Streptomyces Sp. VN1, a Producer of Diverse Metabolites Including Non
    www.nature.com/scientificreports OPEN Streptomyces sp. VN1, a producer of diverse metabolites including non-natural furan-type anticancer compound Hue Thi Nguyen1, Anaya Raj Pokhrel 1, Chung Thanh Nguyen1, Van Thuy Thi Pham1, Dipesh Dhakal 1, Haet Nim Lim1, Hye Jin Jung1,2, Tae-Su Kim1, Tokutaro Yamaguchi 1,2 & Jae Kyung Sohng1,2* Streptomyces sp. VN1 was isolated from the coastal region of Phu Yen Province (central Viet Nam). Morphological, physiological, and whole genome phylogenetic analyses suggested that strain Streptomyces sp. VN1 belonged to genus Streptomyces. Whole genome sequencing analysis showed its genome was 8,341,703 base pairs in length with GC content of 72.5%. Diverse metabolites, including cinnamamide, spirotetronate antibiotic lobophorin A, diketopiperazines cyclo-L-proline- L-tyrosine, and a unique furan-type compound were isolated from Streptomyces sp. VN1. Structures of these compounds were studied by HR-Q-TOF ESI/MS/MS and 2D NMR analyses. Bioassay-guided purifcation yielded a furan-type compound which exhibited in vitro anticancer activity against AGS, HCT116, A375M, U87MG, and A549 cell lines with IC50 values of 40.5, 123.7, 84.67, 50, and 58.64 µM, respectively. In silico genome analysis of the isolated Streptomyces sp. VN1 contained 34 gene clusters responsible for the biosynthesis of known and/or novel secondary metabolites, including diferent types of terpene, T1PKS, T2PKS, T3PKS, NRPS, and hybrid PKS-NRPS. Genome mining with HR-Q-TOF ESI/MS/MS analysis of the crude extract confrmed the biosynthesis of lobophorin analogs. This study indicates that Streptomyces sp. VN1 is a promising strain for biosynthesis of novel natural products.
    [Show full text]
  • Differential Gene Expression Analysis of Aclacinomycin Producing Streptomyces Galilaeus ATCC 31615 and Its Mutant
    Differential gene expression analysis of aclacinomycin producing Streptomyces galilaeus ATCC 31615 and its mutant Master’s thesis MD A B M Sharifuzzaman Department of Life Technologies Molecular Systems Biology University of Turku Supervisor: Co-supervisor: Professor Mikko Metsä-Ketelä, Ph.D. Keith Yamada, M.Sc. Department of Life Technologies PhD candidate Antibiotic Biosynthesis Engineering Lab Department of Life Technologies University of Turku Antibiotic Biosynthesis Engineering Lab University of Turku Master’s thesis 2021 The originality of this thesis has been checked in accordance with the University of Turku Quality assurance system using the Turnitin Originality check service Summary Streptomyces from the genus Actinomycetales are soil bacteria known to have a complex secondary metabolism that is extensively regulated by environmental and genetic factors. Consequently they produce antibiotics that are unnecessary for their growth but are used as a defense mechanism to dispel cohabiting microorganisms. In addition to other isoforms Streptomyces galilaeus ATCC 31615 (WT) produces aclacinomycin A (Acl A), whereas its mutant strain HO42 (MT) is an overproducer of Acl B. Acl A is an anthracycline clinically approved for cancer chemotherapy and used in Japan and China. A better understanding of the how the different isoforms of Acl are made and investigations into a possible Acl recycling system would allow us to use metabolic engineering for the generation of a strain that produces higher quantities of Acl A with a clean production profile. In this study, RNA-Seq data from the WT, and MT strains on the 1st (D1), 2nd (D2), 3rd (D3), and 4th (D4) day of their growth was used and differentially expressed genes (DEGs) were identified.
    [Show full text]