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