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

SCREENING FOR ANTIBIOTICS FROM INDIGENOUS STREPTOMYCETES, THEIR GENETIC AND MUTATIONAL ANALYSIS

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”.

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

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

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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

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

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

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 ion chromatogram of crude extract CRF1 102 Figure 4.10b Selected ion chromatogram of extract CRF11 102 Figure 4.11a Total ion chromatogram of crude extract CRF14 103 Figure 4.11b Selected ion chromatogram of extract CRF14 103 Figure 4.12a Total ion chromatogram of crude extract CRF17 104 Figure 4.12b Selected ion chromatogram of extract CRF17 104 Figure 4.13a Total ion chromatogram of crude extract SCF18 105 Figure 4.13b Selected ion chromatogram of extract SCF18 105 Figure 4.14a Total ion chromatogram of crude extract SCF25 106 Figure 4.14b Selected ion chromatogram of extract SCF25 106 Figure 4.15a Total ion chromatogram of crude extract SCF31 107 Figure 4.15b Selected ion chromatogram of extract SCF31 107 Figure 4.16a Total ion chromatogram of crude extract CTF9 108 Figure 4.16b Selected ion chromatogram of extract CTF9 108 Figure 4.17a Total ion chromatogram of crude extract CTF13 109

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Figure 4.17b Selected ion chromatogram of extract CTF13 109 Figure 4.18a Total ion chromatogram of crude extract CTF15 110 Figure 4.18b Selected ion chromatogram of extract CTF15 110 Figure 4.19a Total ion chromatogram of crude extract CTF24 111 Figure 4.19b Selected ion chromatogram of extract CTF24 111 Figure 4.20a Total ion chromatogram of crude extract CTF25. 112 Figure 4.20b Selected ion chromatogram of extract CTF25 112 Figure 5.1 Working up scheme of the terresterial Streptomyces sp. 119 RSF18 Figure 5.2 1H NMR (DMSO, 300 MHz) of Genenthiocin (1) 121 Figure 5.3 1H NMR (DMSO, 300 MHz) of Val-genenthiocin (2) 121 Figure 5.4 13C NMR (DMSO, 125 MHz) of Genenthiocin (1) 122 Figure 5.5 13C NMR (DMSO, 125 MHz) of Val-genenthiocin (2) 122 Figure 5.6 Molecular structure of the cyclic thiopeptides geninthiocin 122 (1) and val- geninthiocin (2) Figure 5.7 CID-MS/MS spectrum of val-geninthiocin (2); 125 fragmentation of [M+H]+ measured on a quadrupole ion trap instrument Figure 5.8 Selected CID-MS/MS key fragments of the [M+H]+ 125 precursor ions of geninthiocin (1, R = OH) and Val- geninthiocin (2, R = H) obtained on a quadrupole ion trap mass spectrometer 1 Figure 5.9 H NMR (CDCl3, 300 MHz) of chalcomycin (3) 128 13 Figure 5.10 C NMR (CDCl3, 125 MHz) of chalcomycin (3) 128 Figure 5.11 Molecular structure of chalcomycin 128 Figure 5.12 Working up scheme of the Streptomyces sp. CRF 14 131 1 Figure 5.13 H NMR (CDCl3, 300 MHz) of Alborexin (4) 131 13 Figure 5.14 C NMR (CDCl3, 125 MHz) of Alborexin (4) 132 Figure 5.15 Molecular structure of Alborexin (4) 132 Figure 5.16 Working up scheme of the Streptomyces sp. BG-5 134 1 Figure 5.17 H NMR (CDCl3, 300 MHz) of Ochromycenone (5) 134 13 Figure 5.18 C NMR (CDCl3, 125 MHz) of Ochromycinone (5) 135 Figure 5.19 Molecular structure of Ochromycinone (5) 135 1 Figure 5.20 H NMR (CDCl3, 300 MHz) of Emycin D (6) 135 13 Figure 5.21 C NMR (CDCl3, 125 MHz) of Emycin D (6) 136 Figure 5.22 Molecular structure of Emycin D (6) 136 Figure 5.23 Molecular structure of 1-Acetyl-β-carbolin (7) 136 Figure 5.24 Working up scheme of the Streptomyces sp. CTF 9 138 1 Figure 5.25 H NMR (CDCl3, 300 MHz) of Phenyl acetic acid (8) 138 13 Figure 5.26 C NMR (CDCl3, 125 MHz) of Phenyl acetic acid (8) 139 Figure 5.27 Molecular structure of phenyl acetic acid (8) 139 1 Figure 5.28 H NMR (CDCl3, 300 MHz) of β-indole-lactic acid (9) 140 Figure 5.29 Molecular structure of β-indole-lactic acid (9) 140 Figure 5.30 Working up scheme of the Streptomyces sp. CTF-15 142 1 Figure 5.31 H NMR (CDCl3/MeOH, 300 MHz) of resistomycin(10) 142 Figure 5.32 Molecular structure of resistomycin (10) 142

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1 Figure 5.33 H NMR (CDCl3/MeOH, 300 MHz) of tetracenomycin 144 Figure 5.34 Molecular structure of Tetracenomycin D (11) 144 Figure 5.35 1H NMR (MeOH, 300 MHz) of actinomycin D (12) 145 Figure 5.36 Molecular structure of Actinomycin D (12) 145 Figure 5.37 Working up scheme of the Streptomyces sp. SCF 25 147 Figure 5.38 1H NMR (MeOH, 300 MHz) of tryptophol (13) 147 Figure 5.39 Molecular structure of Tryptophol (13) 148 Figure 5.40 1H NMR (MeOH, 300 MHz) of tyrosol and ferulic acid 148 Figure 5.41 Mol structure of Tyrosol (14) 148 Figure 5.42 Mol structure of Ferulic acid (15) 148 Figure 6.1 The 16s rRNA gene sequence of the isolate RSF17 158 Figure 6.2 An example electropharogram of the 16s rRNA gene 159 sequence of the isolate RSF17 Figure 6.3 Phylogenetic Tree of Streptomyces sp. Strain RSF17 160 Figure 6.4 Phylogenetic Tree of Streptomyces sp. Strain RSF18 160 Figure 6.5 Phylogenetic Tree of Streptomyces chromofuscus Strain 161 RSF23 Figure 6.6 Phylogenetic Tree of Streptomyces matensis Strain BG5 161 Figure 6.7 Phylogenetic Tree of Streptomyces macrosporeus Strain 161 CRF1 Figure 6.8 Phylogenetic Tree of Strain Streptomyces vinaceus Strain 162 CRF2 Figure 6.9 Phylogenetic Tree of Streptomyces heliomycini Strain 162 CRF11 Figure 6.10 Phylogenetic Tree of Streptomyces pulcher Strain CRF17 162 Figure 6.11 Phylogenetic Tree of Streptomyces griseoincarnatus 163 Strain SCF18 Figure 6.12 Phylogenetic Tree of Streptomyces macrosporeus Strain 163 SCF25 Figure 6.13 Phylogenetic Tree of Streptomyces malachitofuscus Strain 163 SCF31 Figure 6.14 Phylogenetic Tree of Streptomyces malachitofuscus Strain 164 CTF9 Figure 6.15 Phylogenetic Tree of Streptomyces erythrogriseus Strain 164 CTF13 Figure 6.16 Phylogenetic Tree of Streptomyces labedae Strain CTF14 164 Figure 6.17 Phylogenetic Tree of Streptomyces griseoincarnatus strain 165 CTF15 Figure 6.18 Phylogenetic Tree of Streptomyces rochei Strain CTF20 165 Figure 6.19 Phylogenetic Tree of Streptomyces macrosporeus strain 165 CTF25 Figure 7.1 Comparison of the effect of different media compositions 177 and culture conditions on the yield and antimicrobial activity of the compounds produced by strain RSF18 Figure 7.2 Comparison of the effect of different media compositions 178 and culture conditions on the yield and antimicrobial

activity of the compounds produced by strain CTF9 Figure 8.1 Selected chloramphenicol resistant mutants of wild strain 200 RSF18 obtained by different mutagenic treatments exhibiting variations in morphologic characters Figure 8.2 Chloramphenicol resistant mutants of wild strain RSF18 201 exhibiting variations in colony characteristics obtained by treatments with different concentrations of MNNG. Figure 8.3 Variations in antimicrobial activity of the 202 Chloramphenicol resistant mutants of parental strain Streptomyces sp. RSF18 obtained by different mutagenic treatments. Figure 8.4 Enhanced antimicrobial activity of finally selected mutants 203 against Bacillus subtilus in comparison with the parental strain RSF18 Figure 8.5 Number of chloremphenicol resistant mutants obtained by 205 treatment with different concentrations of ehtidium bromide Figure 8.6 Number of chloremphenicol resistant mutants obtained by 207 treatment with different concentrations of MNNG Figure 8.7 Number of chloremphenicol resistant mutants obtained by 208 UV irradiation for different time intervals. Figure 8.8 %age increase in the antimicrobial activity of the parental 209 strain RSF18 by different mutagenic treatments Figure 9.1 Similarity among the isolates on the basis of different 213 physiological characteristics including production of melanin, utilization of different sugars as carbon source, utilization of organic acids, utilization of oxalates, hydrolysis of urea and hemolysis, the cluster analysis was performed by MINITAB version11. Figure 9.2 Dendrogram obtained using 16s rRNA gene sequences of 213 the selected Streptomyces strains showing clustering by the unweighted pair group method with arithmetic means (UPGMA). Figure 9.3 Antimicrobial activity pattern of the selected Streptomyces 215 isolates Figure 9.4 Cytotoxicity of the selected Streptomyces isolates 215 determined by brine shrimp(Artimia salina) microwell cytotoxity assay Figure 9.5 Comparison of the isolates on the basis of the number of 218 metabolites produced by each isolate as detected in HPLC- MS analysis Figure 9.6 Frequencies of the mutants of parental strain RSF18 222 exhibiting variable antimicrobial activity, obtained by different mutagenic treatments

Summary

SUMMARY

The 30 soil samples were collected from different sites of the province Punjab, including saline agricultural farmlands, rose cultivated fields at university campus and northern areas of Pakistan. The soil samples were treated chemically and physically for the enrichment and selective isolation of streptomycetes. On the whole 110 active Streptomyces strains were isolated from these soil samples using selective media and stringent culture conditions. The isolates including RSF17, RSF18, RSF23, BG5, CRF1, CRF2, CRF11, CRF14, CRF17, SCF18, SCF25, SCF31, CTF9, CTF13, CTF14, CTF15, CTF20, CTF23, CTF24 and CTF25 were selected as the representative strains on the basis of their origin and antimicrobial activity for detailed study. The morphological, microscopic, biochemical and physiological characterization of the selected strains strongly suggested that all the isolates are the members of the genus Streptomyces. In prescreening studies, the selected strains were cultivated on small scale (as 1 liter shaking culture), the resulting culture broth of each of the strain was freeze dried and the residue was extracted with ethyl acetate to obtain the crude extracts. The crude extracts of individual strains were screened biologically and chemically, in biological screening the activity of the extracts was determined against a set of test organisms including, Bacillus subtilus, Staphylococcus aureus, Streptomyces viridochromogen TU 57, E coli, Candida albicans, Mucor mehimi, Chlorella vulgaris, Chlorella sorokiana and Scendesmus subspicatus. Additionally a cytotoxicity test was performed using brine shrimp (Artimia salina) microwell cytotoxicity assay. Antimicrobial activities against almost all the test organisms were observed, however the most promising activities were exhibited by the isolates, RSF17, RSF18, BG5, CRF17, SCF18, SCF25, CTF9, CTF13, CTF15 and CTF20. The maximum cytotoxicity i.e. more than 90% mortality of the shrimp larvae was exhibited by the isolates, RSF23, CRF2, CRF14, SCF25, CTF14 and CTF15. In chemical screening the crude extracts obtained from the strains were analyzed by TLC (Thin Layer Chromatography) and HPLC-MS. The TLC plates were developed with a

CH2Cl2/5% MeOH solvent system. Zones on the developed plates were visualized under UV light (at 254nm and 366nm) and the components showing UV absorbance or fluorescence were marked. Later the TLC plates were sprayed with anisaldehyde/sulfuric acid or Ehrlich’s reagent (detection of indoles) for the further localization of interesting xi

Summary zones. The colored bands (metabolic finger print) produced by the reaction between spray reagent and the metabolites were marked and documented by scanning. In HPLC-MS analysis the measured masses in each of the crude extract were searched in the AntiBase (a data base of the microbial secondary metabolites) and the number of hits for each of the measured mass (i.e. the number of possible compounds for a particular mass in the data base) was determined. The higher number of hits in the database for a particular mass suggested that the respective compound may be very common and small number of hits or 0 hits for a particular mass in the data base suggested that the respective compound may be a unique or rare metabolite and may have not been isolated from the microbes previously. On the basis of the metabolic profiles (metabolic fingerprint) obtained by chemical screening and the biological activities exhibited by each of the selected strain, six Streptomyces strain were selected as the competent strains and were cultivated on larger scale (20-50 liter) for the identification of the antimicrobial compounds produced by them. In preparative screening the strains were cultivated as shaking cultures on an orbital shaker or in lab fermenters at 28ºC for 5-7 days. In each case after harvesting, the culture broth was passed through a filter press to separate the cell mass from the liquid phase, the resulting mycellial cake was extracted with ethyl acetate and acetone, while the liquid phase was passed through the XAD resin and later the resin was extracted with methanol. The solvents containing the crude extracts were evaporated on a rotavapour and the few grams of dry crude extract of each of the strain were obtained. The crude extract of each strain was fractionated on a silica ge l column and the fractions were purified by preparative TLC, reverse phase silica gel columns and gel exclusion chromatography etc. The pure fractions obtained were identified by mass spectrometry (EI-MS, ESI-MS, ESIHR-MS) and NMR spectroscopy (1H NMR and 13C NMR) along with data base searches (AntiBase, DNP etc). The strain RSF18 cultivated as 15 liter shaking culture, yielded three pure compounds including, the cyclic thiopeptides Geninthiocin and Val geninthiocin (new) and the macrolide chalcomycin. The strain CRF17 cultivated in a 20 liter lab fermenter (Biostat E) yielded one pure compound, the Alborexin (a polyether compound). The strain BG5 cultivated as 25 liter shaking culture yielded three pure compounds that were identified as, Ochromycinone, Emycin D and 1-acetyl-β carbolin. The strain CTF9 was cultivated in a fifty litre

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Summary fermenter (Biostat U), isolation and purification resulted in many pure fractions however most of the fractions were found to be unstable and only two fraction were identified including phenyl acetic acid and β-indole lactic acid. The strain CTF15 was cultivated as twenty five litre shaking culture, the extraction and purification of this culture broth yeilded three compounds including Resistomycin, Actinomycin D and Tetracenomycin D. Similarly the strain SCF25 was grown in fifty litre fermenter (Biostat U), extraction and purification of this culture broth yeilded many fractions which were difficult to purify and were unstable, however three fractions were identified from this strain including Tryptophol, Ferulic acid and Tyrosol. The final taxonomic status of the interesting strains (seventeen strains), selected on the basis of the preescreening and preparative screening results was determined by 16S rRNA gene sequencing. In 16S rRNA gene sequence studies these indigenous isolates exhibited genetic similarity up to 99% with different species of the genus Streptomyces. In cultural optimization studies the strains RSF18 and CTF9 were investigated under six different media compositions, A, B, C, D, E, F and four different setes of culture conditions, CC1, CC2, CC3, and CC4, to get the maximum yeild of the metabolites produced by them. In mutational analysis, the effect of four mutagens including two chemical (Ethidium bromide and N- methyl N- nitro- N- nitrosoguanidine or MNNG) and two physical mutagens (short wavelength UV and Gamma rays) was studied on the growth and antimicrobial activity of the strain RSF18. The mutants with up to 57% increase in the antimicrobial activity as compared to the wild strain were obtained from the 500 μg and 750 μg MNNG treatments.

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Introduction

CHAPTER 1 INTRODUCTION

The extensive use of antibiotics since their introduction into the medical practices make the utilization of existing antimicrobial agents to be rapidly eroding, shifting the balance in favor of multi drug resistant pathogens, almost one third to one half of antibiotics prescriptions are counted unnecessary (Drlica, 2001). There are four major mechanisms that mediate bacterial resistance to drugs. Bacteria produce enzymes that inactivate the drugs e.g. beta lactamases can inactivate penicillins and cepalosporins by cleaving the beta lactam ring of the drug (Ohmori et al., 1977). Bacteria synthesize modified targets against which the drug has no effect e.g. a mutant protein in the 30s ribosomal subunit can result in resistance to streptomycin and a methylated 23s rRNA can result in resistance to erythromycin. Bacteria decrease their permeability such that an effective intracellular concentration of the drug is not achieved e.g. changes in porins can reduce the amount of penicillin entering the bacterium (Lindmark et al., 2004). Bacteria actively export drugs using a “multidrug resistance pumps” (MDR pump). The MDR pump imports protons and, in an exchange-type reaction, exports a variety of foreign molecules including certain antibiotics, such as quinolineas. Besides drug resistance also occurs due to genetic changes in the organism, either a chromosomal mutation or the acquisition of a plasmid or transposon (Martinez and Baquero, 2000; Kummerer, 2004). The resistance of numerous pathogenic bacteria and fungi to commonly used bioactive secondary metabolites is presently an urgent focus of research, and new antifungal and antibacterial molecules are necessary to combat these pathogens (Fgiura et al., 2005). Screening of microorganisms for the production of novel antibiotics has been intensively pursued for many years by scientists. Antibiotics have been used in many fields including agriculture, veterinary and pharmaceutical industry.

Developmental History of Antibiotics

The most widely accepted concept defines an antibiotic as “ the low-molecular weight (MW < 2000 Dalton) secondary metabolites from natural sources including their chemically or biosynthetically produced derivatives, which show inhibition of the growth of higher organisms (e.g. tumour cells) or pathogens (e.g. bacteria, fungi, viruses) at low

Introduction concentration, and subsequently can be used to cure the infectious diseases” (Berdy, 2005). The history and development of antibiotics as therapeutic agents are very similar to the patterns for other types of drugs. Relatively ineffective attempts to use materials, which are now recognized as having antibiotics association, can be detected in folk medicine and the prepenicillin scientific literature. Reports some dating back 2500 hundred years indicate that various ancient and primitive peoples applied moldy bread, soybean cured and other materials to boils and wounds liable to infection; this can be considered as a folk medicine type of antibiotics therapy (Barton and Nakanishi, 1999). During the 1880’s attempts were made to utilize antagonism to achieve an ecologic control of the human microbial flora by introducing selected non-pathogenic organisms. Pyocyanase, a crude mixture of metabolites extracted from Pseudomonas aeruginosa, became available around the turn of the century and could be considered as the first commercial antibiotics (Crueger and Crueger, 2003). Establishment of the therapeutic feasibility of penicillin antagonism in the early 1940s stimulated the intensive efforts that culminated in the high level of current antibiotic development. During the second world war, the demand for chemotherapeutic agents to treat wound infections led to the development of a production process for penicillin and the beginning of the era of antibiotics research. Intensive screening programs in all industrial countries continue to increase the number of described antibiotics: notwithstanding the failures, the almost exponential increasing of the total number of discovered compounds in the last decades surprisingly became constant. In 1940 only 10-20, in 1950 300-400, in 1960 approximately 800-1000 and in 1970 already 2500 antibiotics were known. From that time the total number of known bioactive microbial metabolites has doubled in every ten years. In 1980 about 5000, in 1990 10000 and in 2000 already almost 20000 antibiotic compounds were known. By the end of 2002 over 22000 bioactive secondary metabolites (including antibiotics) were published in the scientific and patent literature (Berdy, 2005).

Major Groups of Microorganisms Producing Antibiotics

Antibiotics and similar natural products, being secondary metabolites can be produced by almost all types of living things. They are produced by prokaryotic (Prokaryotae, Monera) and eukaryotic organisms belonging to the Plant and Animal Kingdom. The

Introduction secondary metabolite producing ability, however, is very uneven in the species of living world. Among the microorganisms, three major microbial groups, bacteria, actinomycetes and fungi are involved in the production of antibiotics (Table 1.1). In fungi, only the antibiotics produced by the aspergillaceae and moniliales are of practical importance. (Crueger and Crueger, 2003). In the bacteria there are many taxonomic groups which produce antibiotics. The greatest variety in structure and number of antibiotics is found in the actinomycetes (Fig 1.1), especially in the genus streptomycetes. A compilation of microbial sources of antibiotics, discovered in United States and Japan reveals that approximately 85% are produced by actinomycetes, 11% by fungi, and 4.5% by bacteria. Berdy (2005) provided the statistical overview of bioactive metabolites produced by different groups of microorganisms (Fig 1.1). Table: 1.1 Approximate number of bioactive microbial metabolites according to their producers and bioactivities (Berdy, 2005) Bioactive secondary microbial metabolites Source Antibiotics Bioactive metabolites Total (With other No antibiotic (Antibiotics plus Total bioactive activity) activity “Other activities”) metabolites Bacteria 2900 (780) 900 (1680) 3800 Eubacteriales 2170 (570) 560 (1150) 2750 Bacillus sp. 795 (235) 65 (300) 860 Pseudomonas sp. 610 (185) 185 (370) 795 Myxobacter 400 (130) 10 (140) 410 Cyanobacter 300 (80) 340 (420) 640 Actinomycetales 8700 (2400) 1400 (3800) 10100 Streptomyces sp. 6550 (1920) 1060 (3000) 7630 Rare actinos 2250 (580) 220 (800) 2470 Fungi 4900 (2300) 3700 (6000) 8600 Microscopic fungi 3770 (2070) 2680 (4750) 6450 Penicillium/aspergillus 1000 (450) 950 (1400) 1950 Basidiomycetes 1050 (200) 950 (1150) 2000 Yeasts 105 (35) 35 (70) 140 Slime moulds 30 (5) 20 (25) 60 Total microbial 16500 (5500) 6000 (11500) 22500 The numbers of antibiotics (without other activities), the numbers of the antibiotics exhibiting additional “other” bioactivities (in parenthesis), and the “other bioactive” metabolites as well as the total numbers are summarized according to the main producer types and several specific producer species.

Introduction

In Fig. 1.1 the total number of discovered antibiotics is summarized chronologically from 1950 to 2000 in five years intervals, indicating their main sources, such as Streptomyces, rare actinomycetes, fungi and bacteria (1a), as well as their percentages indicated in the same periods (1b).

Figure 1.1 Distribution of the discovered antibiotics according to their origin (Berdy, 2005).

Introduction

Antibiotics production could be of ecological significance for the life of an organism in nature, as secondary metabolites antibiotics serve regulatory role during differentiation, perhaps acting as temporary inhibitory agents. To date, most new antibiotics have been detected strictly by empirical screening methods, with little attention to their possible roles for the producing strains. Bu’Lock (1961) borrowed the term “secondary metabolite” from plant physiology and applied it to microbiology. The primary metabolites are essential for life and reproduction of cells, the primary metabolism functions similarly in all microorganisms, however following is true for secondary metabolites: every secondary metabolite is formed by only a few organisms, secondary metabolites are seemingly not essential for growth and reproduction, their formation is extremely dependent on environmental condition (Trossell, 1993). The regulation of the biosynthesis of secondary metabolites differs significantly from that of the primary metabolites. Zahner (1979) suggested, besides the five phases of the cells own metabolism (intermediary metabolism, regulation transport, differentiation and morphogenesis), secondary metabolism is considered a “playing field” for the evolution of further biochemical development, which can proceed without damaging primary metabolism. Genetic changes leading to the modifications of metabolites would be expected not to have any major effect on normal cell function. If a genetic change leads to the formation of a compound that in some way is beneficial, then this genetic change would be fixed in the cell’s genome, perhaps becoming essential. In this way the former secondary metabolite would be converted in to a primary metabolite (Motta et al., 2004).

The Actinomycetes (Major Producers of Antibiotics)

Actinomycetes are a group of morphologically diverse, Gram positive bacteria having in common DNA with unusually high GC content in the range of 63-78% (Stackebrandt and Woese, 1981; Goodfellow and Cross, 1984; Embley and Stackebrandt, 1994; Madigan and Martinko, 2005). The morphological diversity ranges from micrococci which divide as cocci to members of the actinobacteria that form pleomorphic rods or go through coccus rod coccus life cycle, more complex still are the branched filament forming actinomycetes. In most of the cases, the successful growth and branching form a network of filaments called a mycelium, which morphologically resemble to the mycelium formed

Introduction by the filamentous fungi. Most of the actinomycetes form spores, the manner of spore formation and spore morphology varies in different genera and are used as important criteria in the taxonomic grouping of actinomycetes. Phylogenetically, the filamentous actinomycetes form a coherent group, thus, the mycelial and spore forming habit is of both phylogenetic and taxonomic importance. Concepts of taxonomy and natural relationships of these bacteria have been revised radically in recent years by results of partial sequence analysis of 16s rRNA (Stackenbrandt and woese, 1981). These analysis show the actinomycetes constitute a major subdivision of the Gram-positive eubacteria in an evolutionary line phylogenetically distinct from the bacillus-Lactobacillus- Streptococcus group. The actinomycetes are usually divided into seven major groups, including actinobacteria, nocardioforms, actinoplanetes, thermomonospora, maduromycetes, streptomycetes and a group with multilocular sporangia. There are over 100 validly described actinomycetes genera but not all have been assigned to families (Goodfellow, 1989).

Characteristics of Streptomycetes

Among the actinimycetes, the members of genus Streptomyces are most extensively exploited organisms for the production of antibiotics and other related health care products. Their natural habitat soil is nutritionally, biologically and physically complex and variable, demanding for their fast adaptation. As a consequence, they are able to perform a broad range of metabolic processes, and produce an immense diversity of bioactive secondary metabolites (Ball et al., 1989; Wachinger et al., 1989). Members of the streptomycetes are ubiquitous, and there is no doubt that soil is the natural habitat where they find suitable conditions for growth and proliferation, however there are many other “biotopes” which may be contaminated or infected by soil, the most important of these are fodder and other organic materials such as grains, decaying wood etc. Along with the fresh water, marine habitats are also being explored for the isolation of bioactive Streptomyces, especially marine Streptomyces have proved themselves as a good source of novel antibiotics (Blunt et al., 2003; Poumale et al., 2006). Streptomycetes are defined to be the organisms producing an extensively branching primary or substrate mycelium as well as a more or less abundant secondary or aerial

Introduction mycelium. The aerial mycelium shows characteristic modes of branching and in the course of the streptomycete life cycle, these hyphae are partly transformed into chains of spores, which are often called conidia or arthrospores. The life cycle of streptomycetes comprises different stages including: substrate mycelia, aerial hyphae and chains of spores (Kelemen et al., 1998; Champness, 2000). Under favourable conditions, one or two germ tubes emerge from a spore and grow by tip extension and branch formation to give rise to a substrate mycelium. After about 2-3 days, aerial hyphae grow up in a process that involves the action of a large number of bld genes (bldA, B, C, D, G, H, I, K etc). When aerial growth stops, multiple septa subdivide the apical compartment into unigenomic pre-spore compartments. These subsequently change in shape; wall thickening occurs and grey spore pigment is deposited, to generate dessiccation-resistant spores. Sporulation and septation depends on six regulatory loci (whiA, B, G, H, I and J), which are also needed for expression of at least one (sigF) of two late regulatory loci, whiD and sigF; these in turn play important roles in the thickening of the spore wall and the deposition of the grey spore pigment (spore pigment is specified by the whiE gene cluster). The timing of each stage and their switches are subtly controlled by a large variety of regulatory genes, induced or repressed upon extracellular signals. Studies of the two groups of S. coelicolor A3(2) developmental mutants: bld mutants (failing to produce the aerial mycelia), and whi mutants (aerial hyphae fail to complete the production of the normal grey spores), indicate a high complexity of regulatory network which includes morphogenesis and production of secondary metabolites (Fig 1.2) (Kieser et al., 2000; Chater, 2001). Streptomycetes are chemoorganotrophic, non fastidious microorganisms that need only an organic source (as a carbon source), an inorganic nitrogen source and some mineral salts for growth. Growth factor requirements are rare, and a wide variety of carbon sources, such as sugars, alcohols, organic acids, amino acids, and some aromatic compounds, can be utilized. Most isolates produce extracellular hydrolytic enzymes that permit utilization of polysaccharides including starch, cellulose, hemicelluloses etc, proteins, fats and some strains can use hydrocarbons, lignin, tannin or even rubber.

Introduction

Figure 1.2 The life cycle of Streptomyces coelicolor A3(2) (Kieser et al., 2000).

The following criteria may be employed for the characterization of an unknown streptomycetes isolate, morphology, microscopic appearance, biochemistry, physiology, patterns of enzymes and proteins and molecular genetics. Streptomyces display a striking number of macroscopic features such as, colours of aerial mycelium, substrate mycelium and pigment diffusion into the medium, consistency of mycelium and colony morphology. The biochemical properties that have been found to be use full for the separation of streptomycetes from other genera of actinomycetes include, LL- diaminopemelic acid, no characteristic pattern of sugars, a typical pattern of fatty acids, no mycolic acid and a typical pattern of menaquinones. Various physiological tests used in early studies on Streptomyces have been found to be rather unreliable and of little taxonomic value. Only two of them have been employed in the International

Introduction

Streptomyces Project (ISP) (Shirling and Gottlieb, 1966), the formation of melanin pigment and the utilization of nine carbon sources. However taxonomists continued to characterize their cultures by numerous tests and there is no doubt that several physiological properties are very significant for the characterization of Streptomyces (William et al., 1983; Kutzner et al., 1986). The most commonly used criteria for physiological testing include, determination of temperature range for growth, pH, formation of melanin, utilization of carbohydrate and similar compounds as carbon source, utilization of organic acids, formation of organic acids, use of nitrogen sources, reduction of nitrate to nitrite, hydrolysis of esculin and arbutin, hydrolysis of urea. Few investigations have also been made into the taxonomic value of serological properties and restriction of total DNA i.e. restriction fragment length polymorphism (RFLP) for actinomycetes (Ridal and William, 1983). Another approach for the determination of Phylogenetic relationships between different species is the application of DNA-DNA homology. Organisms belonging to one species are expected to exhibit a high degree of homology of their genetic material (Yamaguchi, 1967). Additionally 16S rRNA sequence data have proven invaluable in streptomycetes systematics, in which they have been used to identify several newly isolated streptomycetes (Fguira et al., 2005). The most striking property of Streptomyces is the extent to which they produce antibiotics. They play an important role in medicine, agriculture and biotechnology. These bacteria produce about 85% of commercially and medically useful antibiotics (Miyadoh, 1993) and approximately 60% of antibiotics that have been developed for agricultural use (Tanaka and Ômura, 1993). In some studies it has been seen that more than 50% of all the isolated strains have been proved to be antibiotic producers. Some organisms produce more than one antibiotic, and often the several kinds produced by one organism are not even chemically related. The antibiotics produced by streptomycetes show a wide diversity of chemical structures including, aminoglycosides, ansamycins, anthracyclines, β-lactams, macrolides, peptides, polyenes, polyethers, tetracyclines and other antibiotics outside from these defined structural classes. Table 1.2 includes some of the famous commercially produced antibiotics, which belong to these structural classes and are produced by Streptomyces.

Introduction

Table 1.2 Some common antibiotics produced by species of Streptomyces (Walsh, 2003). Structural class Antibiotic Producer Activity/Mode of action Aminoglycosides Streptomycin S. griseus Antibacterial agents, Kanamycin A, B S. kanamyceticus Some are broad spectrum, Hygromycin B S. hygroscopicus Inhibit protein synthesis Neomycin S. fradiae

Tetracyclines Tetracycline S. aurofaciens Antibacterial, broad Chlorotetracycline S. aurofaciens spectrum, Inhibit protein synthesis Macrolides Avermectins S. avermitilis Antibacterial, inhibit Erythromycin S. erythrius protein synthesis Clindamycin S. lincolnesis β-Lactams Cephamycin A, B S. chartreusis Antibacterial, inhibit cell Clavulanic acid S. clavuligerus wall synthesis Polyenes Nystatin S. noursei Antifungal Amphotericin B S. nodosus Peptides/ Neocarzinostatin S. carzinostaticus Antitumor agents Glycopeptide Bleomycin A2, B2 S. verticillus Undefined Chloremphenicol S. venezuelae Antibacterial, broad spectrum

In respect to the application of streptomycetes for many purposes, genetic analysis proved to be a very useful tool for a better understanding of the biology of these organisms like, the arrangement of genes for differentiation, knowledge of genes for the biosynthesis of antibiotics, improvement of antibiotic producing strains, creation of hybrid antibiotics, discovery of genome rearrangements, detection of insertion sequences, and development of protoplast techniques. Furthermore, genetic studies contributed to the understanding of phenotypic variations. The recently published complete genome sequence of the model strain Streptomyces coelicolor A3(2) revealed several more remarkable features and at the same time opened new questions (Bentley et al., 2002).

Introduction

The 8.6 Mb linear chromosome belongs to one of the largest completely sequenced bacterial genome. Its replication is bidirectional, proceeding from the centrally located oriC to the chromosome termini (Zakrzewska and Schrempf, 1992). The terminal ends are replicated by so called “end patching”, where DNA synthesis is primed by terminal proteins (Bao and Cohen, 2001). The coding density is largely uniform across the chromosome, which contains remarkably 7825 predicted genes far more than the genome of Escherichia coli (4289 genes) or Sacharomyces cerevisiae (6203 genes). The genome also shows a strong emphasis on genes coding for putative regulators (12.3%) and 65 sigma factors. The ability to exploit a large variety of nutrients is supported with the 10.5% of potentially secreted proteins, since the majority of them are hydrolases (proteases/peptidases, chitinases, celullases and amylases). The distribution of known different types of genes discloses however a central core spanning about the half of the chromosome and a pair of arms. In this biphasic division nearly all important genes (like for cell division, DNA replication, amino acid biosynthesis etc.) are located in the stable central core, while the genes for the secondary metabolism and hydrolytic enzymes lie in the arms. From 18 clusters that may be involved in the secondary metabolism, only three are related to antibiotic synthesis, whereas the others seem to be responsible for the biosynthesis of the “stress metabolites” like hopanoides, siderophores and others (Bentley et al., 2002). In streptomycetes as the aerial hyphae appear and sporulation occurs, the production of secondary metabolites is induced (Hopwood, 1988). It is thought that the morphological and physiological differentiation and the onset of secondary metabolite production result from common elements of regulation (Takano et al., 1992; Bibb, 1996). Studies have shown that mutants unable to sporulate (bald mutants) are also defective in regulation of production secondary metabolites (Chater and Hopwood, 1989; Chater and Bibb, 1996; Pope et al., 1996). S. coelicolor A3(2) produces at least four secondary metabolites: actinorhodin, undecylprodigiosin, A-factor and methylenomycin (Chater and Hopwood, 1989). Actinorhodin is a blue-pigmented dimeric isochromanequinone which is synthesized from an acetyl-CoA starter unit and seven malonyl-CoA extender units (Allman et al., 1981; Strohl and Connors, 1992). It is one of the best studied antibiotics with respect to its biosynthetic pathway and to its pleiotropic regulation (Allman et al.,

Introduction

1981; Malpartida and Hopwood, 1984; Doull and Vining, 1990; Horinouchi and Beppu, 1992; Hutchinson et al., 1993; Fernandez-Moreno et al., 1994). Amycolatopsis methanolica belongs to the family of Pseudonocardiaceae (Embley et al., 1988; Embley and Stackebrandt, 1994). It is one of the few methanol-utilizing Gram-positive bacteria, employing the ribulose monophosphate (RuMP) cycle of formaldehyde fixation (fructose bisphosphate aldolase cleavage variant) (Hazeu et al., 1983). Because of its characteristics, this organism is a good candidate for fermentative production of aromatic amino acids and secondary metabolites derived from them (Dijkhuizen et al., 1985; De Boer, 1990). In recent years the main studies with this organism concerned the detailed characterization of the pathways involved in aromatic amino acid biosynthesis, methanol utilization, and the development of suitable plasmid vectors and of transformation systems (Dijkhuizen et al., 1993; Euverink, 1995; Vrijbloed, 1996). Secondary metabolites are synthesized from primary metabolite precursors. Studies of primary metabolism in these types of organisms have been very limited but would provide important information about the switch between primary and secondary metabolism and for further improvement of industrial processes for the production of secondary metabolites.

Antibiotics Screening

The search for bioactive compounds in nature is a multi-step procedure, which begins with the selection of suitable sources and appropriate test systems. In a general screening strategy, crude extracts are evaluated by chemical, biological and pharmaceutical screening approaches; the latter can focus on looking for bioactive substances and often provides the advantages of greater sensitivity and sample throughput for the industrial High-throughput-Screening (HTS) (Grabley and Thiericke, 1999). However, novel compounds, which may be active against other targets, are overlooked. To overcome this problem, Zahner and many other researchers started systematically a chemical screening of crude extracts in the 1980s (Zähner et al., 1982). The chromatographic characteristics of metabolites on thin layer chromatography (TLC) plates, as well as their chemical reactivity towards staining reagents under defined reaction conditions, allows visualizing of an almost complete fingerprint of the secondary metabolite pattern (Grabley and

Introduction

Thiericke, 1999). The use of this method has led to the isolation of nearly all metabolites of a given strain, and the various unknown compounds can then be biologically tested in pure state. Due to the recent increase of the sensitivity of mass spectroscopy (MS), nuclear magnetic resonance (NMR) instruments, and the rapidly growing chemical databases (AntiBase, DNP, CA), allows the dereplication of known compounds and their structure determination. Presently the screening is conducted in combination with high- performance liquid chromatography (HPLC), ultraviolet (UV), HPLC-DAAD, HPLC- CD, HPLC-MS, HPLC-NMR-MS or GC-MS systems (Dobler et al., 2002; Malthum et al., 1999).

Partial Identification and Dereplication

It is obvious that despite of the existence of modern methods, the isolation and structural elucidation of natural compounds is a time-consuming and expensive process. The dereplication is an important step with the aim to distinguish between known compounds and unknowns, and consequently allowing to exclude the known compounds at an earlier stage. The principle of this method is to compare data fragments of mixtures or pure metabolites with suitable literature data. This might be carried out by comparing the UV

(Fiedler, 1993) or MS data and HPLC retention times with appropriate reference data collections. This method needs only negligible sample amounts and affords reliable results, if authentic samples had been available to measure the reference data. UV data and MS fragmentation patterns are also useful to identify unknown metabolites, if these show similar chromophores or fragmentation patterns as known analogues. First results have shown that already known natural products can be identified easily even from crude extracts obtained from bacterial broths. Application of these methods is a very valuable tool to make the process of finding new biological and pharmacological active compounds more efficient. As it will never be possible to collect a complete sample set and to measure all experimental data under identical conditions, reference values from the literature have to be used. If NMR data are selected, results from 1D measurement can be translated into substructures, which then will be used for a database search. In this case, normally sufficiently pure samples are required. Databases with the NMR or UV data and a variety of other molecular descriptors can be searched using computers

Introduction

(Bukingham and Thompson 1997). The most comprehensive data collection of natural compounds is the Dictionary of Natural Products (DNP), which compiles metabolites from all natural sources, including plants. The data collection “AntiBase” (Laatsch, AntiBase 2007) is, however, more appropriate for the dereplication of microbial products, as the identification depending on structural features and spectroscopic data is more comprehensive, faster and more reliable. In the case of new compounds, a database search is also helpful because novel skeletons are rare and usually related compounds are already known which are easily revealed by a database search, thus identifying at least the compound class. Finally, the Chemical Abstracts (CA), the most comprehensive bank of information worldwide, is used for a final confirmation that a given structure is new. Sub-structure searches with small fragments are not possible here for technical reasons. The combination of liquid chromatography with detection methods such as NMR spectroscopy (HPLC NMR) and tandem mass spectrometry (HPLC-MS/MS) has recently led to new strategies by which biological matrices, e.g., crude plant extracts (Bringmann et al., 2001) or extracts from bacterial culture broths (Bringmann and Lang, 2003), are screened to obtain as much information as possible about known constituents even with a minimum amount of material. As most compounds of interest are thermally labile, HPLC-ESI MS/MS would be the method of choice to identify known molecules from multi-component mixtures with high selectivity and sensitivity (Oka et al., 2004). The absolute configuration of the pure components can be confirmed by application of circular dichroism (CD) spectroscopy (Bringmann and Busemann, 1998).

Approaches to Get New Microbial Metabolites

Natural product resources, including the microbial world, are mainly unexplored both in its dimension and in the respect of geographic, ecological and environmental points of view. There surely exist, besides the presumed numbers of microorganisms, millions of microbes in the environment that are presently untouchable for the science. The DNA community analysis demonstrates the presence of much more novel organisms in the environmental samples, than those was originally believed, which are not detectable and cannot be isolated by “classical” methods. Unique ecosystems as it is supposed may result in unique organisms with unique metabolic pathways. The isolation of microbes

Introduction from diverse ecosystems especially from under-represented sites (extreme circumstances, sea, etc.) may result in promising, new, until now unexplored producers. There is a big potential in the screening of the simply unknown species. The hardly detectable and almost unculturable actinomycetales species, their genetic exploitation and metabolic expression give also almost unlimited possibilities. The application of new molecular techniques can greatly improve the effectivity of the exploration of uncommon, unreported groups of various microbes, first of all new actinomycetales strains, greatly increasing the chemical diversity. To increase the screening efficacy for secondary metabolites of bacteria, microbiologists developed a polymerase chain reaction (PCR)- based screening assay for genes e.g. Polyketide synthases, non-ribosomal polypeptide synthases (NRPSs), dNDP-glucose dehydratases and halogenases (Martens et al., 2002). The problem of this approach is, however, that these genes occur very frequently, but may not be expressed (silent genes). In recent years, genes of e.g. polyketide synthases were located and isolated, in accordance, genetically manipulated microorganisms can be used to generate new metabolites by realigning the synthetic capacities of different species. Novel natural products are optimized based on their biological activities to yield effective chemotherapeutic and other bioactive agents (Bechthold et al., 1999). Additionally biotransformation techniques can be used to investigate new natural products. This method enables derivatization of known compounds by esterfication, reduction, oxidation, demethylation, or glycosylation utilizing the enzymes of living microorganisms. For example, glycopeptides, antitumor metabolites from Streptomyces verticillus (Fujii et al., 1973) were found to be good antibiotics or antineoplastic agents, e.g. bleomycin A1 (Oshtari et al., 1997). Bleomycins are currently used for clinical treatment of Hodkin’s lymphoma, carcinomas of the skin, head, and neck, and tumors of testis (Oshtari et al., 1997). Some antibiotics derived from natural resources need to be transformed structurally in order to optimize their pharmacological properties. In order to be successful, screening must be an interdisciplinary activity, combining the activities of microbiology, chemistry, biotechnology, medicine and bibliographic. Microbiology is involved in the isolation and identification of microorganisms, strain preservation, and testing for biological activity, while biotechnology deals with fermentation practices, cultural optimizations and scale up. The analytical procedures as

Introduction well as the approaches to purification of biologically intersiting molecules fall under the purview of the chemist. The bibliographic specialist searches the literature and is responsible for data handling. Figure 1.3 represents an integrated approach between research groups in microbiology, natural product chemistry, biotechnology, and medicine to discover the metabolic capabilities of bacteria for the production of bioactive compounds.

Microbiology Isolation of Bacteria, characterization, phylogenetic studies, culture preservation, Biological Screening (antimicrobial activities, cytotoxic studies etc)

Medicine Biotechnology/ Genomics Clinical trials, cell line Scale up, media optimization, strain inhibition etc improvement, Fermentation etc

Chemistry Chemical screening, isolation and purification of compounds, structure elucidation etc

Figure 1.3 Integrated approaches to explore the metabolic capabilities of bacteria for the production of bioactive compounds.

Mutagenic Strain Improvement

Wild strains frequently produce a mixture of chemically closely related substances. Mutants which synthesize one component as the main product are preferable, since they make possible a simplified process for product recovery. There are several genetics and molecular genetics methods useful for the improvement of secondary metabolite production. They include the: Classical random chemical mutagenesis Mutagenesis through radiations Rational selection of spontaneous or induced mutations 16

Introduction

Transposition mutagenesis Targeted deletions and duplications Genetic recombination by protoplast fusion Random chemically induced mutagenesis and fermentation screening continues to be an effective way to increase the productivity of fermentation processes (Baltz, 1999). The main advantages of random mutagenesis are that high levels of mutagenesis can be achieved by certain chemical mutagens, and that mutagenesis can be carried out with little or no knowledge of what mutations would be beneficial. Mutant screening operations can be manual or highly automated (Queener and Lively, 1986; Vinci and Byng, 1999). A shortcoming of random mutagenesis is that most effective chemical mutagens induce a very limited spectrum of base-pair substitutions (Baltz, 2000). A variety of chemicals are known which are mutagenic, and these may be classified into three groups according to their mode of action. Mutagens which effect nonreplicating DNA, Base analogs: which are incorporated into replicating DNA due to their structural similarity with one of the naturally occurring bases, and frameshift mutagens: which intercalate into DNA during replication or repair and cause insertion or deletion of one or few nucleotide pairs. The most potent mutagen for actinomycetes is, N-methyl-N'-nitro- N-nitrosoguanidine (MNNG), its use in a mutation program is difficult because of its carcinogenic effects, but it is one of the most effective chemical mutagen. A large proportion of mutants are found under optimal conditions with a low killing rate. In Streptomyces coelicolar, for instance, 8-10% of the survivors are found to be auxotrophs, and in Escherichia coli up to 50% of the surviving population consists of mutants (Coulondre and Miller, 1977). Ninety percent of the mutations induced by MNNG are GC to AT transitions, a small extant deletions and frameshift mutation are also found as a result of the deletions of the GC pairs (Baltz and Stonesifer, 1985a, 1985b; Baltz 1986). Both ultraviolet radiations and ionization radiations are used in mutagenesis studies. The mechanisms of mutagenesis are quite different for each type of radiation. The most effective mutagenic agent is short wavelength UV in the range of 200-300 nm with an optimum of 254nm (Harold and Hopwood, 1970). The most important effect of short wavelength UV is the formation of dimmers between adjacent pyrimidines or between pyrimidines of complimentary strands, which results in cross linking. UV radiations also

Introduction induce transition of GC to AT transversions, frameshift mutations and sometime deletions. Long wavelength UV radiations in the range of 300-400 nm has less lethal effects, however if the exposure of cells is carried out in the presence of various dyes which interact with DNA, greater death rates and increased mutation frequency results. Ionizing radiations like gamma rays act by causing ionization of the medium through which they pass. Single stranded and double stranded breaks occurs with a significantly higher probability than other mutagens. The single stranded breaks are usually repaired by repair mechanisms; however double stranded breaks results in major structural changes, such as translocation and inversion etc. Besides an optimal mutagenesis, the method of mutant selection is crucial for the effective screening of mutants. There are basically two ways of screening. A direct random selection of survivors from a mutagenized population can be examined for antibiotic production. Direct selection for mutants that express increased secondary metabolite production has at least three important advantages. First, nearly 100% of the strains selected can have single mutations potentially influencing product yields. Secondly, strains containing rare mutations can be isolated and screened with appropriate selections. Thirdly, mutations that arise by spontaneous mutation can be screened. A limitation of mutant selections is that only a fraction of potential mutations that increase product yields will be subject to selection (Baltz, 2001). The second basic method for mutant selection is the selective isolation of mutants. In this procedure a high cell density of a mutagenized population can be plated on a selective medium containing a concentration of a toxic substance that prevents the wild type from growing, only the resistant clones can develop. In this way, mutants may be isolated which are resistant to antibiotics or antimetabolites. Additionally the mutagenized cell density can also be searched for auxotrophs in a selective mutant isolation strategy.

Aims of the Study

The main goal of the present study was the thorough investigation of indigenous streptomycetes from different localities in our area, Punjab (Pakistan), for the production of useful/interesting bioactive secondary metabolites. The objectives also included the study of the biodiversity of streptomycetes in this region and specifically to scrutinize the

Introduction chemical diversity of the metabolites produced by these isolates. The following lines summarize the more specific objectives of the study.

Isolation of indigenous streptomycetes exhibiting antimicrobial activities from their natural habitat especially from soil. Characterization and identification of the isolates exhibiting antimicrobial activity and their taxonomic grouping. Biological and chemical screening of the isolated strains and selection of competent strains. Cultivation and fermentation of the selected strains on pilot scale for the production of active metabolites. Isolation, purification and structure elucidation of active antimicrobial compounds from the culture broth of the selected strains. Cultural optimization of the selected strains to get the maximum yield of the interesting metabolites. Determination of the effect of different chemical and radiation mutagens on the ability of the selected strains to produce interesting metabolites.

Materials and Methods

CHATER-2 MATERIALS AND METHODS

MATERIALS All media and solutions were prepared in glass-distilled water and were autoclaved at 121 C at 15 lb/inch2 for 15 minutes. Glassware was washed properly and oven dried before use. All the chemicals used are of analytical grade.

Table 2.1 ACTINOMYCETES ISOLATION AGAR (Difco Laboratories)

S. No. COMPONENTS gm L-1 1 Glycerol 5.0 2 Sodium propionate 4.0 3 Sodium caseinate 2.0 4 Asparagine 0.1 5 K2HPO4 0.5 6 MgSO4.7H2O 0.1 7 FeSO4.7H2O 0.001 8 Agar 15 pH adjusted to 7.8

Table 2.2 GLYCEROL-CASEIN-KNO3 AGAR (Kuster and Williams, 1964) S. No. COMPONENTS gm L-1 1 Glycerol 10.0 2 KNO3 2.0 3 Casein 0.3 4 NaCl 2.0 5 K2HPO4 2.0 6 MgSO4.7H2O 0.05 7 CaCO3 0.02 8 FeSO4.7H2O 0.01 9 Agar 18 pH adjusted to 7.8

Materials and Methods

Table 2.3 CHITIN AGAR (Hsu and Lockwood, 1975)

S. No. COMPONENTS gm L-1 1 Chitin (colloidal) 4.0 2 KH2PO4 0.3 3 K2HPO4 0.7 4 MgSO4.7H2O 0.5 5 FeSO4.7H2O 0.01 6 ZnSO4.7H2O 0.001 7 MnCl2.4H2O 0.001 8 Agar 20 pH adjusted to 7.8

Table 2.4 GLYCEROL-ARGININE AGAR (El-Nakeeb and Lechevalier, 1963)

S. No. COMPONENTS gm L-1 1 Glycerol 12.5 2 Arginine 1.0 3 NaCl 1.0 4 K2HPO4 1.0 5 MgSO4.7H2O 0.5 6 Fe2(SO4)3.6H2O 0.01 7 CuSO4.5H2O 0.001 8 ZnSO4.7H2O 0.001 9 MnSO4.H2O 0.001 10 Agar 15 pH adjusted to 7.8

Table 2.5 GLUCOSE-YEAST EXTRACT-MALT EXTRACT (GYM) BROTH (Shirling and Gottlieb, 1966)

S. No. COMPONENTS gm L-1 1 Malt extract 10 g 2 Glucose 4 g 3 Yeast extract 4 g pH was adjusted to 7.8 using 2N NaOH.

Materials and Methods

Table 2.6 GLUCOSE-YEAST EXTRACT-MALT EXTRACT (GYM) AGAR (Shirling and Gottlieb, 1966)

S. No. COMPONENTS gm L-1 1 Malt extract 10 g 2 Glucose 4 g 3 Yeast extract 4 g 4 Agar 12 pH was adjusted to 7.8 using 2N NaOH.

Table 2.7 INORGANIC SALTS-STARCH AGAR (Shirling and Gottlieb, 1966)

S. No. COMPONENTS gm L-1 1 Starch 10.0 2 (NH4)2SO4 2.0 3 K2HPO4 1.0 4 MgSO4.7H2O 1.0 5 NaCl 1.0 6 CaCO3 2.0 7 Trace salts sol 1.0 ml 8 Agar 12 pH adjusted to 7.4

Table 2.8 GLYCEROL-ASPARAGINE AGAR (Shirling and Gottlieb, 1966)

S. No. COMPONENTS gm L-1 1 Glycerol 10.0 2 L-Asparagine 1.0 3 K2HPO4 1.0 4 Trace salts sol or (SPV-4) 1.0 ml 5 Agar 12 pH adjusted to 7.4

Table 2.9 TRACE SALTS SOLUTION

S. No. COMPONENTS gm /100 ml

1 FeSO4.7H2O 0.1 2 ZnSO4.7H2O 0.1 3 MnCl2.4H2O 0.1

Materials and Methods

Table 2.10 TRACE ELEMENTS SOLUTION SPV-4 (Voelskow, 1988)

S. No. COMPONENTS gm L-1

1 CaCl2 .2H2O 4.0 2 Fe (III) citrate 1.0 3 CuSO4.5H2O 0.04 4 MnSO4 0.2 6 ZnCl2 0.1 7 CoCl2 0.022 8 Na2MoO4.2H2O 0.025 9 Na2B4O7.10H2O 0.1

Table 2.11 MEDIUM FOR MELANIN FORMATION (Suter, 1978)

S. No. COMPONENTS gm L-1 1 Glycerol 15 2 L-Arginine.HCl 5.0 3 L-Tyrosine 1.0 4 L-Methionine 0.3 5 K2HPO4 0.5 6 MgSO4.7H2O 0.2 7 FeSO4.7H2O 0.01 8 CuSO4.5H2O 0.01 9 CaCl2 .2H2O 0.01 10 ZnSO4.7H2O 0.01 11 MnSO4.4H2O 0.04 12 Agar 15 pH adjusted to 7.4

Table 2.12 BASAL MEDIUM FOR UTILIZATION OF SUGARS (Shirling and Gottlieb, 1966)

S. No. COMPONENTS gm L-1

1 (NH4)2SO4 2.64 2 KH2PO4 2.38 3 K2HPO4.3H2O 5.56 4 MgSO4.7H2O 1.0 5 FeSO4.7H2O 1.10 6 CuSO4.5H2O 6.46 7 ZnSO4.7H2O 1.50 8 MnCl2.4H2O 7.90 9 Agar 15 pH adjusted to 7.0

Materials and Methods

Table 2.13 BASAL MEDIUM FOR UTILIZATION OF ORGANIC ACIDS (Cowan and Steel, 1965) S. No. COMPONENTS gm L-1

1 (NH4)2SO4 1.0 2 KH2PO4 0.27 3 Na2HPO4.2H2O 0.53 4 MgSO4.7H2O 0.5 5 NaCl 2.0 6 Bromothymol blue 0.025 7 Agar 12 pH adjusted to 7.0

Table 2.14 SALTS OF ORGANIC ACIDS

S. No. COMPONENTS gm L-1 1 Potassium gluconate 5.0 2 Trisodium citrate 2.0 3 Sodium malate 5.0 4 Sodium lactate 5.0 5 Sodium malonate 3.0

Table 2.15 UTILIZATION OF OXALATES

S. No. COMPONENTS gm L-1 1 Potassium oxalate 1.0 2 (NH4)2HPO4 1.0 3 KH2PO4 0.5 4 MgSO4.7H2O 0.2 5 NaCl 1.0 6 Agar 12 pH adjusted to 7.0 Table 2.16 MEDIUM FOR HYDROLYSIS OF UREA

S. No. COMPONENTS gm L-1 1 Glucose 1.000 2 Casein-peptone 1.000 3 KH2PO4 1.510 4 Na2HPO4.2H2O 1.980 5 MgSO4.7H2O 0.500 6 NaCl 5.000 7 Phenol red 0.012 8 Agar 12.00 pH adjusted to 6.8

Materials and Methods

Table 2.17 SOLUTION OF UREA

S. No. COMPONENTS gm/100ml 1 Urea 10 2 Distilled Water (up to) 100 ml

Table 2.18 BASAL MEDIUM FOR HEMOLYSIS

S. No. COMPONENTS gm L-1 1 Casein-peptone 10.0 2 Meat extract 10.0 3 NaCl 5.0 4 Agar 12.0 pH adjusted to 6.8

Table 2.19 L - BROTH (Gerhardt et al., 1994)

S. No. COMPONENTS gm L-1 1 Tryptone 10.0 2 Yeast extract 5.0 3 NaCl 5.0 pH adjusted to 7.0

Table 2.20 L - AGAR (Gerhardt et al., 1994)

S. No. COMPONENTS gm L-1 1 Tryptone 10.0 2 Yeast extract 5.0 3 NaCl 5.0 4 Agar 12.0 pH adjusted to 7.0

Table 2.21 NUTRIENT-BROTH (Gerhardt et al., 1994)

S. No. COMPONENTS gm L-1 1 Peptone 5.0 2 Beef extract 3.0 pH adjusted to 7.0

Materials and Methods

Table 2.22 NUTRIENT-AGAR (Gerhardt et al., 1994)

S. No. COMPONENTS gm L-1 1 Peptone 5.0 2 Beef extract 3.0 3 Agar 12.0 pH adjusted to 7.0

Table 2.23 MULLAR HINTON AGAR (Barry and Effinger, 1974)

S. No. COMPONENTS gm L-1 1 Beef Extract 2 2 Acid Casein hydrolysate 17.5 3 Starch 1.5 4 Agar 17.0 pH adjusted to 7.3

Table 2.24 MEDIUM FOR STREAK TEST (Williams et al., 1983)

S. No. COMPONENTS gm L-1 1 D-Glucose 15.0 2 Glycerol 2.5 3 Soybean meal 15.0 4 Yeast extract 1.0 5 NaCl 5.0 6 CaCO3 1.0 7 Agar 15.0 pH was adjusted to 6.8 using

Table 2.25 SOJA-MANNIT MEDIUM

S. No. COMPONENTS gm L-1 1 Soybean meal (defatted) 20 g 2 D(-)-Mannit 20 g 3 Agar. 18 g pH was adjusted to 7.8 using 2N NaOH.

Materials and Methods

Table 2.26 SABOURAUD-AGAR (for test organism Candida albicans)

S. No. COMPONENTS gm L-1 1 Glucose 40 2 Peptone 10 3 Agar 20 4 Demineralised water 1000 ml pH was adjusted to 7.8 using 2N NaOH.

Table 2.27 Fe-EDTA

S. No. COMPONENTS gm100 ml-1

1 FeSO4.7 H2O 0.7 2 EDTA (Titriplex III) 0.93 g 3 Demineralized water 100 ml

0.7 g of FeSO4.7 H2O and 0.93 g EDTA (Titriplex III) are dissolved in 80 ml of demineralised water at 60 °C and then diluted to 100 ml.

TRACE ELEMENT SOLUTION II:

Table 2.28 SOLUTION A:

S. No. COMPONENTS mg/10 ml

1 MnSO4.H2O 16.9 2 Na2MoO4.2H2O 13.0 3 Co(NO3)2.6H2O 10.0 4 Demineralized water 10.0 ml

Table 2.29 SOLUTION B:

S. No. COMPONENTS mg/10 ml

1 CuSO4.5H2O 5.0 2 H3BO3 10.0 3 ZnSO4.7H2O 10.0 4 Demineralized water 10.0 ml Solutions A was added to B and diluted to 100 ml with demineralized water.

Materials and Methods

Table 2.30 BOLD’S BASAL MEDIUM (BBM) (Kantz and Bold, 1969)

S. No. COMPONENTS gm L-1

1 NaNO3 0.250 g 2 KH2PO4 0.175 g 3 K2HPO4 0.075 g 4 MgSO4.7 H2O 0.075 g 5 NaCl 0.025 g 6 CaCl2.2 H2O 0.025 g 7 Fe-EDTA 1.0 ml 8 Trace element solution II 0.1 ml

Salts are dissolved in 10 ml of demineralised water and added to Fe-EDTA and trace element solution II. The mixture made to one litre with demineralised water. Solid medium was prepared by adding 18 g of bacto agar.

Table 2.31 A: M2 MEDIUM

S. No. COMPONENTS gm L-1 1 Malt Extract 10 2 Yeast extract 4 3 Glucose 4 4 Dimineralized H2O (up to) 1 L

Table 2.32 B: SM MEDIUM

S. No. COMPONENTS gm L-1 1 Soya been fat 20 2 Manitol 20 3 Demineralized water (up to) 1 L

Table 2.33 C: FLEISCH EXTRACT MEDIUM

S. No. COMPONENTS gm L-1 1 Glucose anhydrous 10 2 Peptone 2 3 Yeast extract 1 4 Meat extract 1 5 Demineralized water (up to) 1 L

Materials and Methods

Table 2.34 D: CaCl2 MEDIUM

S. No. COMPONENTS gm L-1 1 Yeast Extract 40 2 Glucose 5 3 CaCl2 45 4 Demineralized water 1 L

Table 2.35 E: LB MEDIUM

S. No. COMPONENTS gm L-1 1 Trypton 10 2 Yeast extract 5 3 NaCl 10 4 Glucose 5 5 Demineralized water 1 L

Table 2.36 F: FISH MEDIUM (Juliana et al., 2007)

S. No. COMPONENTS gm L-1 1 Glucose 21 2 Fish flour 5 3 Flour 10 4 MgSO4 0.5 5 NaCl 1 6 CaCl2 0.5 7 Trace elements 10 ml

Table 2.37 TRACE ELEMENTS (STOCK SOLUTION) FOR FISH MEDIUM

S. No. COMPONENTS gm L-1

1 FeSO4 0.2g 2 COCl2 0.04g 3 CaCl2 0.04g 4 Manganese chloride, 0.04g 5 Zinc sulfate 0.08g 6 Sodium borat 0.08g

Materials and Methods

Table 2.38 TE (TRIS EDTA) BUFFER

S. No. COMPONENTS gm L-1 1. Tris base 1.211 2. EDTA 0.5M 20 ml 3 Distilled H2O Up to 1 L 1.211 gm of Tris base was dissolved in 600 ml Distilled water and the pH was adjusted to 8.0 with HCl , later 20 ml 0.5 M EDTA was mixed and volume was made up to 1 liter.

Table 2.39 0.5 M EDTA

S. No. COMPONENTS gm L-1 1 EDTA 186.12 2 Distilled H2O (Up to) 1 L

Table 2.40 LYSOZYME SOL (50 mg/ml)

S. No. COMPONENTS mg /ml 1 Lysozyme 50

Table 2.41 20% SODIUM DODICYLE SULFATE (SDS) SOL

S. No. COMPONENTS g/100 ml 1 Sodium Dodisyle Sulfate 20

Table 2.42 PROTEINASE K (20mg/ml)

S. No. COMPONENTS mg/ml 1 Proteinase K 20

Table 2.43 PHENOL, CHLOROFORM, ISOPROPANOL SOL (25:24:1)

S. No. COMPONENTS ml/50ml 1 Phenol 25 2 Chloroform 24 3 Isopropanol 1

Materials and Methods

Table 2.44 70% ETHANOL

S. No. COMPONENTS ml/100ml 1 Ethanol 70 2 Demineralized H2O 30

Table 2.45 10 X TBE (TRIS BASE BORIC ACID EDTA) (Sambrook and Russell, 2001)

S. No. COMPONENTS gm L-1 1 Tris base 108.0 2 Boric acid 55.0 3 Sodium EDTA 9.25

Table 2.46 RUNNING BUFFER (0.5 X TBE)

S. No. COMPONENTS ml L-1 1 10 X TBE 50 ml 2 Distilled water 950 ml

Table 2.47 ETHIDIUM BROMIDE SOLUTION

S. No. COMPONENTS mg /ml 1 Ethidium bromide 10 2 Distilled water 1 ml The ethidium bromide was mixed in water and filtered before use

Table 2.48 6X LOADING DYE

S. No. COMPONENTS 1 Tris-HCl 10 mM 2 Bromothymol blue 0.03% 3 Xylene cyanol 0.03% 4 Glycerol 60% 5 EDTA 60 mM

Materials and Methods

Table 2.49 0.9 % AGAROSE

S. No. COMPONENTS gm/100ml 1 Agarose 0.9 2 TBE 0.5X (up to) 100 ml

Agarose was dissolved in 0.5 X TBE and heated to melt in water bath and 2.5 l (5 mg ml-1) of Ethidium bromide was added in it before pouring.

Table 2.50 PRIMERS Primers for16S rRNA Gene

Forward Primer (P1) 5′-AGAGTTTGATCATGGC-3′ Reverse Primer (P2) 5′-TACCTTGTTACGACTT-3′

Table 2.51 PCR CONDITIONS

S. No. STEPS CONDITIONS 1 Denaturation 94˚C for 5 minutes 2 Denaturation 94˚C for 1 minute 3 Annealing 55˚C for 1 minute 4 Extension 72˚C for 2 minute 5 Final extension 72˚C for 5 minutes 6 No. of cycles 30 7 Hold at 4˚C

Table 2.52 COMPONENTS OF REACTION MIXTURE In a sterile amplification tube, the PCR reaction mixture components were added as: S.No Item µL /50 µL 1 10 p mol Forward Primer (P1) 2 2 10 p mol Reverse Primer (P2) 2 3 dNTPs 1 4 Pfu buffer 5 5 Pfu DNA Polymerase 1 6 Template DNA 1 7 Deionized water 38

Materials and Methods

DNA polymerase used

Pfu DNA polymerase (native) 2.5 u/µL,(Fermentas # EPO 571)

Buffer used in PCR mix

10X Pfu Buffer with MgSO4 0.6 ml (Fermentas Lot # 00000705) Gel Extraction Kits used

PrepEase TM Gel Extraction Kit (Product # 78757, 250 preps)

QIAquick Gel Extraction Kit (Cat. # 28704, Qiagen, Inc.)

Table 2.53 STOCK SOLUTION FOR ANISALDEHYDE S. No. COMPONENTS ml L-1 1 Methanol 850 2 Acetic acid 140 3 Sulphuric acid 10 1 ml anisaldehyde was added to 100 ml of a stock solution

Table 2.54 STOCK SOLUTION FOR EHRLICH’S REAGENT S. No. COMPONENTS ml L-1 1 Methanol 750 2 Hydrochloric acid 37% 250 1gm 4-dimethylaminobenzaldehyde was dissolved in 100 ml of stock solution it gives red colouration with indol and yellow for other N-heterocycles.

Table 2.55 NINHYDRIN S. No. COMPONENTS ml L-1 1 Iso-propanol 950 2 Collidin (2,4,6-trimethylpyridin) 25 3 Acetic acid (96%) 25

0.3gm ninhydrin (2, 2- dihydroxyindan-1, 3-dione) was dissolved in 95 ml iso-propanol. The mixture was added to 5 ml collidin (2, 4, 6-trimethylpyridin) and 5 ml acetic acid (96%). This reagent gave a blue to a violet colouration with amino acids, peptides and polypeptides having free amino groups.

Materials and Methods

Table 2.56 REAGENTS FOR PEPTIDE TEST S. No. COMPONENTS QUANTITY 1 O-dianisidine 0.32 g 2 IN Acetic acid 100 ml 3 Potassium Iodide 0.45 g 4 Na2WO4. 2 H2O 1.5 g 5 KClO3 0.5 g 6 HCL 5 ml 7 Acetone 115 ml 8 Water 10 ml

The reagent was prepared from 100 ml (0.032%) o-dianisidin in 1 N acetic acid, 1.5 g

Na2WO4. 2H2O in 10 ml water, 115 ml acetone and 450 mg KI and stored in dark in a refrigerator. Table 2.57 PALLADIUM (II)-CHLORIDE S. No. COMPONENTS ml L-1 1 Palladium(II)-chloride 5

0.5 g ofPalladium(II)-chloride are dissolved in 100 ml water in presence of few drops of HCl (25 %). It is used to identify sulfur compounds, which give brown/grey colour spots on the TLC after heating. Table 2.58 2N NAOH SOLUTION S. No. COMPONENTS ml L-1 1 NaOH 80

2N NaOH or KOH solutions are used to identify perihydroxyquinones by deepening of the colour from orange to violet or blue. Table 2.59 ANTIFOAMING AGENT S. No. COMPONENTS gm L-1 1 Niax- oil 100 g 2 Etahnol 70% 1000 ml

Table 2.60 2N HCL S. No. COMPONENTS ml L-1 1 HCL 200

Materials and Methods

Table 2.61 ANTIBIOTICS USED FOR DETERMINING SENSITIVITY PATTERN OF THE WILD STRAIN S. No. ANTIBIOTICS Concentration 1 Novobiocin (NV) 5 μg 2 Oxytetracyclin (OT) 30 μg 3 Erythromycin (E) 15 μg 4 Ampicillin (Amp) 25 μg 5 Penicillin (P) 10 μg 6 Carbenicillin (CAR) 100 μg 7 Chloramphenicol (C) 30 μg 8 Gentamycin (CN) 10 μg

Table 2.62 STOCK SOLUTION OF CHLORAMPHENICOL (1mg /ml) S. No. COMPONENTS mg/100ml 1 Chloramphenicol 100 2 Ethanol 100ml

Table 2.63 STOCK SOLUTION OF ETHIDIUM BROMIDE (1mg/ml) S. No. COMPONENTS mg ml-1 1. Ethidium Bromide 10 2 Distilled water 10ml

Table 2.64 STOCK SOLUTION OF N-METHYL-N′-NITRO-N- NITROSOGUANIDINE (MNNG) (1mg/ml) S. No. COMPONENTS mg ml-1 MNNG (N-Methyl-N′-Nitro-N- 1 10 Nitrosoguanidine (MNNG) 2 Distilled water 10ml

Table 2.65 TM BUFFER (Kieser et al. 2000) S. No. COMPONENTS gm/100ml 1 Tris-base 0.605 2 Maleic acid 0.580 3 Distilled water (up to) 100ml pH adjusted to 8.0 ± 0.2

Table 2.66 0.9% SALINE SOLUTION S. No. COMPONENTS gm/100ml 1 NaCl 0.90 2 Water 100ml

Materials and Methods

METHODS

SOIL SAMPLING

Soil samples were collected from different sites including saline agricultural farm lands in the district Jhang (Punjab, Pakistan), Botanical Garden/rose fields of University of the Punjab and northern areas of Pakistan. In actual 30 soil samples were collected from these sites in sterile bags, each sample was collected at the depth of 5-10 cm, the samples were labelled accordingly, quantity for each of the collected sample was 5 grams, different physical factors like pH and temperature of the soil was recorded.

SAMPLE TREATMENT

The soil samples were treated using physical and chemical treatments. In physical treatment the samples were kept at high temperature (50 C) for three weeks, in chemical treatment CaCO3 enrichment methods were used, the soil samples were mixed with

CaCO3 at the ratio of 10:1 and were incubated under moisture rich conditions for seven days at room temperature (Hayakawa et al., 2004).

SELECTIVE ISOLATION OF BIOACTIVE STREPTOMYCES STRAINS

One gram of each soil sample was suspended in 10 ml of sterile water and vortexed for 45 s. The samples were serially diluted and 50 µl of the dilution 10-3 was spreaded over the surface of selective media plates (Table 2.1-2.4) using nystatin and cycloheximide 50µg/ml, as an antifungal agent. After incubation at 28 C for 7-21 days, the actinomycetes colonies were selected and transferred to GYM agar plates (Table 2.6). The actinomycetes cultures were purified by repeated sub culturing on the same medium and other culturing media suplimented with antifungal agents. A preliminary antimicrobial activity test was performed using solid media bioassay method against a standard Gram positive and Gram negative test bacteria. In solid media test, the purified actinomycetes strains were grown for 7 days on plates at 28 C. After the strain has grown properly with well established mycelia, an agar disk was recuperated and shifted to the surface of LB plates covered by 3 ml of top agar containing 50 μl of a 15 hour incubated culture of B. subtilus and E. coli test strains individually, and were incubated overnight at

Materials and Methods

37°C. Only those actinomycetes strains were selected which showed activity against these standard test organisms. The selected strains were characterized morphologically, biochemically, physiologically and genetically (Locci, 1989; Wendisch and Kutzner 1991).

STORAGE OF THE STRAINS

The selected strains were stored in 5 ml aliquots as glycerol stocks (Kirsop and Snell, 1984). Deep-freeze storage in a Dewar vessel, 1’Air liquid type BT 37 A. Capillaries for deep-freeze storage: diameter 1.75 mm, length 80 mm, Hirschmann Laborgerate Eberstadt. Soil for soil culture: Luvos Heilerde LU-VOS JUST GmbH & Co. Friedrichshof (from the health shop).

MACROSCOPIC CHARACTERIZATION

In order to study different macroscopic features of the selected strains a small inoculum of each strain was picked and plated or streaked on GYM-agar plates and was incubated at 28 C for 7 days. The purified colonies obtained were studied for morphologic characteristics including colony size, consistency, shape, elevation, margins, color of aerial/substrate mycelium and pigments diffusing into the medium. The colonies were observed visually and under the microscope (Williams et al. 1989).

MICROSCOPIC CHARACTERIZATION

For cell shape, arrangement of undisturbed hyphae, length of hyphae, branching pattern and chains of arthrospores, 7 days incubated cultures at 28 C were observed under the microscope (Leica 4000 DM). In microscopic analysis it is very important to examine the organism insitu, for this purpose agar plug method of Nishimura and Tawara 1957, was followed. In this method the entire petri dish (or plugs cut out of it and placed on a slide) were subjected to examination.

PHYSIOLOGICAL CHARACTERIZATION

In physiological characterization the two methods employed in International Streptomyces Project (ISP), the formation of melanin and utilization of carbon sources

Materials and Methods

mainly sugars (Shirling and Gottlieb, 1966) was used. However various other physiological tests including, determination of growth temperature range, melanin production, utilization of different carbon sources, utilization of organic acids, utilization of oxalates, hydrolysis of urea, hemolysis test etc as described by Williams et al., 1983 were also performed.

Temperature Range

The first test that should be performed is to determine the optimal growth temperature of an organism. This temperature should be used for further characterization. The selected Actinomycetes strains were inoculated on agar slants (GYM agar, Table 2.6) and were incubated at different temperatures: 10, 20, 28, 37, 40, 45, 50, 55, and 60 C respectively. Growth and formation of aerial mycelium was recorded at the time intervals of 5, 10, and 15 days.

Formation of Melanin

Formation of melanin is indicated by a dark-gray to deep blue- black color in the agar medium after 1-7 days. The results are clear cut (+ or -) and are considered as a very important criteria for differentiation of Streptomyces from other genra of actinomycetes (Held, 1990). Medium used for this test is mentioned in Table 2.11. The agar slants were inoculated with the selected strains and were incubated at 28 C for one week. The agar slants were observed regularly for the formation of melanin pigment. Slants without tyrosine were used as negative control.

Utilization of Sugars as Carbon Source

The following eight different sugars were tested as sole carbon source, D-glucose, D- fructose, L-arabinose, D-glactose, raffinose, D-mannitol, sucrose and mannose. The basal medium used in this test is mentioned in Table 2.12. Following the methods described by Williams et al., 1989 10% solutions of each of these sugars were sterilized by filtration and added to the basal medium (Table 2.12) after autoclaving and cooling to 60 C to give a final concentration of 1%. Agar slants of the complete medium were inoculated and incubated at 28 C for two weeks. The growth was recorded after 7 and 14 days. A slant

Materials and Methods

without carbon source was used as negative control while a slant with glucose as carbon source served as positive control for each strain.

Utilization of Organic Acids

Utilization of organic acids is indicated by alkalization of the medium, the medium changes to a dark blue colour after 6-12 days (Cowan and Steel, 1965). The medium used in this test is mentioned in Table 2.13-2.14, the agar slants of the organic acid utilization medium were prepared and inoculated with the selected strains. The tubes were incubated at 28 C for two weeks. The agar slants of complex medium (GYM medium) was used as control for the quality of inoculums and confluent growth was obtained for each strain. The results were regarded as negative, moderate and strong positive depending upon the colour change of the medium from light blue to dark blue.

Utilization of Oxalate

Oxalate utilization medium is given in Table 2.15, the medium was autoclaved in batches of 92 ml, and before pouring the plates 8ml of 0.1 M solution of CaCl2 was added which resulted in fine precipitates of Ca oxalate. Five organisms were inoculated on each plate in the form of spots and were incubated at 28 C for two weeks. The oxalate utilization was indicated by the appearance of clear zones around the colonies between 5-15 days. The results were recorded as positive or negative.

Hydrolysis of Urea

10% urea solution was prepared and sterilized by filtration, later this solution was mixed with basal medium (Table 2.16-2.17) after autoclaving. The agar slants were inoculated and incubated at appropriate growth conditions. The urease activity was observed by alkalization of the medium as the strain grows on the slant. Ammonia released as a byproduct of the urea change the pH of the medium, as a result yellow color of the medium changes to pink. A control medium without inoculation was kept for comparison of the result. The observations were made daily for five days in order to distinguish

Materials and Methods

between strong and fast positives, moderate positives and negatives. A change in color of the medium from yellow to pink indicated positive result.

Hemolysis

The basal medium used for hemolysis test is mentioned in Table 2.18. The medium was autoclaved in batches of 93 ml and before pouring into plates 7 ml of blood was added in each batch. Each of the agar plate was inoculated in the form of streak by two different strains and was incubated at 28 C for 7 days. The zones of hemolysis were recorded after 2-5 days.

GENETIC CHRACTERIZATION

Isolation of Genomic DNA

High-molecular weight chromosomal DNA of selected Streptomyces strains was prepared from GYM grown mycelia (Felnagle et al., 2007). Approximately 0.5 g (wet weight) of mycelia was washed with 500 µl of TE buffer (Table 2.38). The mycelia were collected again by centrifugation and resuspended in TE buffer along with 20 µl of 50 mg/ml lysozyme. The samples were incubated at 37°C for 4 hours. After incubation 50 µl of 2% (wt/vol) sodium dodecyl sulfate and 5 µl of 20 mg/ml proteinase K were added to each sample, the samples were again incubated at 37°C for 2 to 4 hours. After the second incubation 400 µl of phenol-chloroform-isopropyl alcohol (25:24:1) was added to each of the sample. The samples were subjected to a vortex and then centrifugation at 12,000 rpm for 10 min and the supernatants were transferred to other tubes, this process was repeated twice. In each of the sample supernatant 2volumes of ethanol was added and the samples were kept at -20°C overnight. On next day after centrifugation at 12,000 rpm for 15 min the pellets were washed with 70% ethanol and the DNA was resuspended into 400 µl TE buffer.

PCR Amplification of the 16S rRNA Gene

PCR amplification of the 16S rRNA gene of selected strains was performed using the primers P1and P2 (Table 2.50) as described by Edwards et al. 1989. Approximately 300

Materials and Methods

ng genomic template DNA was used with 150 pmol of each primer per 50 μl reaction volume. Amplification was performed in an automated thermocycler (Perkin Elmer Co. Norwalk- CT, USA) using 1U Pfu DNA polymerase (Fermentas) and Pfu buffer (Fermentas) according to the amplification profile mentioned in Table 2.51. After mixing Primers and other ingredients of PCR (Table 2.52) in amplification tube it was placed in thermoblock of thermocycler, which was fitted with heated lids. The programming was according to the primers used. The conditions of time durations and temperature were optimized and they were specified. The PCR product was analysed by agarose gel electrophoresis and the DNA of the expected size was purified.

Gel Extraction of Amplifiefd DNA Two different kits were used for this purpose.

By PrepEase TM Gel Extraction Kit

The DNA samples to be purified were run over the agarose gel using TBE buffer (Table 2.45-2.46). The DNA samples were stained with ethidium bromide and visualized under the UV illumination. The DNA fragments were excised from the agarose gel by a clean nuclease free razor and the extra gel was removed from the fragments to minimize the volume of the gel. The gel slice containing the DNA fragment was transferred to a clean tube and was weighed. For each 100 mg of agarose gel slice 200 µl of NT buffer (to solubilize the gel) was added to each sample and the samples were incubated at 50°C for 5-10 min until the gel was completely dissolved. The samples were vortexed briefly every 2-3 min to aid the process of gel solubilization. A PrepEaseTM clean up column was placed into a 2 ml PrepEaseTM collecting tube. Each of the samples was loaded directly to the centre of the clean up column and the columns were centrifuged for 1 min at 11,000 rpm. The flow through was discarded and the clean up columns were placed back into the collecting tubes. The column were washed by adding 600 µl NT3 buffer directly to the clean up columns and the columns were centrifuged for 1 min at 11,000 rpm. The flow through was discarded and the clean up columns were again placed back to collecting tubes and again centrifuged for 2 min at 11,000 rpm in order to remove excess NT3 buffer. The clean up columns were placed into 1.5 ml clean appendorrfs and 20 µl of NE buffer was added to each column. The columns were incubated at room temperature for

Materials and Methods

one min in order to increase the yield of eluted DNA. The samples were centrifuged for 1 min at 11,000 rpm, again the 20 µl of NE buffer was added to each column and the columns were incubated at room temperature for one min and were centrifuged for 1 min at 11,000. The eluted contents were concentrated and were stored at -20°C.

QIAquick Gel Extraction Kit (Cat. # 28704, Qiagen, Inc)

After running total PCR product on agarose gels in TBE buffer, the fragment was excised with a clean and sharp scalpel by minimizing the size of the gel slice. After removing extra agarose it was put in a clean microtube. The gel slice was weighed and 3 volumes of buffer “QG” added to 1 volume of gel, by converting the weight of gel (100 mg ~ 100 µL) in to volume, for every mg of gel 3µL volume of buffer QG was added in microtube and incubated it at 50°C for 10 minute (or until the gel slice has completely dissolved), gel was dissolved completely by vortexing the tube every 2–3 minutes during the incubation. After the gel slice dissolved completely, the color of the mixture was checked as to be yellow (similar to buffer QG without dissolved agarose), so as to maintain the optimum pH ≤ 7.5 for DNA binding. The contents were shifted to a QIAquick spin columns while keeping them in a 1.5 ml microtube. The binding of DNA was done after centrifugation for 1 minute at 13000 rpm. The flow-through was discarded and QIAquick columns were placed back in the same microtube. 0.5 ml of buffer QG was added to each QIAquick column and centrifuged for 1 min for the removal of all traces of agarose. The washing was performed by adding 0.75 ml of buffer “PE” to each QIAquick column, the columns were kept standing for 2–5 minutes after addition of PE buffer and were centrifuged for 1 minute at 13000 rpm. The flow-through was discarded and the QIAquick columns were again centrifuged for an additional 1 minute at 13,000 rpm, so that the residual ethanol from buffer PE should be completely removed. The QIAquick columns were placed into a clean 1.5 ml microcentrifuge tube. 50 µL of buffer “EB” (10 mM Tris·Cl, pH 8.5) or H2O was added to the center of each QIAquick column and centrifuged for 1 minute. Alternatively, for increased DNA concentration 30 µL more elution buffer was added to the center of the QIAquick column, the columns were kept standing for 1 minute and then centrifuged for 1 minute at 13000 rpm. The eluted

Materials and Methods

contents were concentrated to approximately 25 µl, the elution was checked on gel by running up to 5 µl. DNA was stored at -20°C till further use.

Sequencing and BLAST Analysis The gene sequencing was done using dye-terminator chemistry with Mega-BACE 1000/4000 DNA automated sequencers (Amersham Bioscience). The obtained 16S rRNA gene sequence data was first analyzed using advance BLAST search program (Altschul et al., 1990) at the NCBI website: http://www.ncbi.nlm.nih.gov/BLAST/. The sequenced data was refined and submitted to Gen Bank, and the accession numbers were obtained.

PRESCREENING

An example of the diversity of Streptomyces metabolism is the observation that individual strains produce different secondary metabolites at the same time. If these metabolites differ fundamentally in structural type and activity spectrum, then there is a risk even with known strains of overlooking other types of natural products by one-sided screening. In order to find out the talented or competent strains from the collection of indigenous Streptomyces, the prescreening approach was adopted. In this strategy a selected strain is cultivated on small scale (1 L shaking culture) and the crude extracts obtained by the extraction of these small size shaking cultures are subjected to biological and chemical screening. This so-called primary screening includes small scale cultivation, extraction, and evaluation of the metabolite pattern by chemical screening and biological assays. The selected strains were first grown as subcultures on agar plates for 3 days using GYM agar medium (Table 2.6). The resulting sub-cultures were then investigated microscopically to confirm the purity of the culture. The subcultures were used to inoculate 4 250 ml of GYM broth (Table 2.5) in 1 L Erlenmeyer flasks (the pH was adjusted to 7.8 before sterilization), the flasks were sterilized and followed by incubation as shaker culture (95 rpm) for 7 days at suitable temperature conditions (28 C). The resulting culture broth was lyophilized (Christ Alpha 2-4 LD), and the residue was extracted with ethyl acetate. Later the crude extracts were subjected to a combinatory of chemical, biological, pharmacological screening and HPLC-MS/MS analysis.

Materials and Methods

BIOLOGICAL SCREENING

In biological screening antimicrobial activity test and cytotoxicity test were performed.

Antimicrobial Activity Test

A standardized filter paper disc agar diffusion procedure is frequently used to determine the drug sensitivity of microorganisms isolated from infectious processes (Dennis, 1974). This method allows for the rapid determination of the efficacy of a drug by measuring the diameter of the zone of inhibition that results from diffusion of the agent into the medium surrounding the disc. In this method filter paper discs of uniform size are impregnated with specified concentrations of different antibiotics and then placed on the surface of an agar plate that has been seeded with the organism to be tested. The medium of choice is poured into plates to a uniform depth and refrigerated for solidification, prior to use the plates are transferred to an incubator at 37 C for 10 to 20 minutes to dry off the moisture that develops on the agar surface. The plates are then heavily inoculated with standardized inoculums by overlaying with a layer of seed agar or by means of a cotton swab to ensure the confluent growth of the organism. The discs are applied aseptically to the surface of agar plates at well spaced intervals. Once applied, each disc is gently touched with a sterile applicator stick to ensure its firm contact with the agar surface. Following the incubation, the plates are examined for the presence of growth inhibition, which is indicated by a clear zone surrounding each disc. The susceptibility of an organism to a drug is determined by the size of this zone, which itself is dependant on various factors such as: (i) the ability and rate of diffusion of the antibiotic into the medium and its interaction with the test organism, (ii) the number of cells of the organism inoculated, (iii) the growth rate of the organism, (iv) the degree of sensitivity of the organism to the antibiotic (Grove and Randall, 1955). The diameter of zone of inhibition is measured in mm. The antimicrobial activity of the crude extracts obtained from selected strains was determined against a set of test organisms including Gram positive bacteria, Gram negative bacteria, fungi and micro algae. The test plates (Petridishes: 94 mm diameter, 16 mm height, Fa. Greiner Labortechnik, Nürtingen), for bacteria were prepared by pouring 14 ml of L-agar (Table 2.20) as base layer; after it has set it was overlayed with 4 ml of the inoculated seed layer. The test plates for fungi were prepared

Materials and Methods

by pouring 14 ml of Sabouraud agar (Table 2.26) as base layer, after it has set it was overlayed with 4 ml of the agar inoculated with fungal spores as seed layer. Bold and basal medium (Kantz and Bold 1969) was used for making algal test plates with the same procedure (Table 2.27-2.30). Paper discs (Antibiotic assay discs: 9 mm diameter, Schleicher & Schüll No. 321 261), were impregnated with 40 µl of crude extract solution

(crude extracts 1µg/µl dissolved in 1:1, CH2CL2/ MeOH); the discs were dried under sterilized conditions so each disc contained 40 µg of crude extract and were placed on the surface of test plates. The preparation of test plates, overlaying of seed layer and application of discs on the surface of test plates was carried out on a clean bench (Laminar-Flow-Box: Kojar KR-125, Reinraumtechnik GmbH, Rielasingen-Worblingen 1). The bacterial and fungal test plates were incubated at 37 C for16- 24 hours, while the algal test plates were incubated at room temperature in daylight or in a photocell (Photo reactor for algal growth: Cylindrical photo reactor (Ø: 45 cm) with ten vertical neon tubes Philips TLD 15 W/25), for 96 hours. After incubation the diameter of zone of inhibition was measured in mm.

Following is the list of test organisms used for biological screening.

1- Staphylococcus aureus 2- Bacillus subtilus

3- Streptomyces viridochromogenes (Tü 57) 4- E.coli

5- Candida albicans 6- Mucor miehei

7- Chlorella sorokiana 8- Chlorella vulgaris

9- Scenedesmus subspicatus 10- Artimia salina (for cytotoxicity)

Brine Shrimp Microwell Cytotoxicity Assay

Cytotoxicity of crude extracts of the selected Actinomycet strains was determined against brine shrimp (Artimia salina). The shrimp lethality assay was proposed by Michael et al., 1956, and later developed by Vanhaecke et al., 1981, and Sleet and Brendel, 1983. It is based on the ability to kill laboratory cultured Artemia nauplii (brine shrimp). The assay is considered a useful tool for preliminary assessment of toxicity (Solís et al., 1993), and it has been used for the detection of fungal toxins (Harwig and Scott, 1971), bacterial

Materials and Methods

metabolites toxicity, plant extract toxicity (McLauglin et al. 1991), heavy metals (Martínez et al., 1998), cyanobacteria toxins (Jaki et al. 1999), pesticides (Barahona and Sánchez, 1999), and cytotoxicity testing of dental materials (Pelka et al., 2000). Test Procedure: Dried eggs of Artemia salina (0.5 g) were added to a 500 ml separating funnel, filled with 400 ml of artificial seawater. The suspension was aerated by bubbling air into the funnel and kept for 24 to 48 hours at room temperature. After aeration had been removed, the suspension was kept for 1 hour undisturbed, whereby the remaining eggs settled down. In order to use only the active larvae, one side of the separating funnel was covered with aluminum foil and the other illuminated with a lamp, whereby the phototropic larvae were gathering at the illuminated side. With a pipette, 30 to 40 shrimp larvae were collected and transferred to a deep-well microtiter plate (wells diameter 1.8 cm, depth 2 cm) filled with 0.2 ml of salt water. The dead larvae were counted (value N), a solution of 20 μg of the crude extract in 5 to 10 μl of DMSO was added and the plate kept at room temperature in the dark. After 24 hours, the number A of the dead animals in each well was counted again under the microscope. The surviving larvae were killed by addition of 0.5 ml methanol so that subsequently the total number G of the animals could be determined. Each test row was accompanied by a blind sample containing pure DMSO instead of a test solution. As a positive control with 100% mortality, actinomycin D (10µg/ml) was used. The mortality rate M was calculated using the following formula:

Where: M = Percent of the dead larvae after 24 hours A = Number of the dead larvae after 24 hours B = Average number of the dead larvae in the blind samples after 24 hours N = Number of the dead larvae before starting of the test G = Total number of larvae

Bioautography Bioautography is a method to localize antibacterial activity on a chromatogram (Botz et al., 2001). In this procedure thin-layer chromatography is coupled with microbiological

Materials and Methods

detection and it has been commonly used for the identification and quantification of several antibiotics. It is considered as a simple, cheap, quite sensitive and specific method to detect a bioactive fraction in a crude extract. Thin layer chromatography of the samples was performed on silica gel plates eluting with a 5% CH2Cl2/ MeOH solvent system. The developed TLC plate was cut into two parts by a clean scissor; one of the half acted as reference TLC and was sprayed with spraying reagent Anisaldehyde/H2SO4, while the second half was fixed over the surface of test plate seeded with test organism in such a way that the upper side of the TLC plate faced the surface of the agar layer. The test plate was incubated at 37 C for 24 hours. Inhibition zones were compared with the Rf of the related spots on the reference TLC plate. Active fractions in a crude extract were marked.

CHEMICAL SCREENING

For chemical screening TLC using some spraying reagents and HPLC-MS methods were used. Chemical screening using thin layer chromatography with various staining reagents and HPLC-MS offers the opportunity to visualize a nearly complete picture of a microbial secondary metabolite pattern (metabolic finger-print). This approach can be used advantageously for both, the detection of so-called "talented" strains, and for qualifying microbial strain collections, especially as a fundamental step of efficiently applied biological high-throughput assays (Antonieta et al., 2006). Based on their metabolic finger-print, microbial isolates can be classified in: (i) non-producing organisms, which gave no indication of the formation of secondary metabolites up to a defined detection limit, (ii) organisms of narrow productivity, which produce one or two secondary metabolites as main products with a restricted dependence to alteration of the culture conditions, and (iii) talented organisms, which are able to synthesize an array of structurally different secondary metabolites.

Thin Layer Chromatography (TLC)

The TLC (thin layer chromatography) is one of the simplest methods used for the detection of the constituents of a crude extract. Secondary metabolites produced under specified culture conditions in appropriate quantity can only be recognized with relative

Materials and Methods

certainty when the thin-layer chromatograms of the various isolated raw products are stained with appropriate spray reagents. Special staining reagents offer the possibility of identifying chosen classes of compounds. By a combination of several reagents which show a broad response to common substance classes, one may obtain an overall picture of the pattern of products from a single strain. The crude extracts obtained from the selected Streptomyces strains were analysed by TLC (thin layer chromatography). In this method a very small drop of a sample was spotted onto the TLC plate (TLC plates: DC- Folien Polygram SIL G/UV254, Macherey-Nagel & Co, Glass plates: Merck silica gel 60 F254, 10× 20 cm) with a capillary and dried, the spotting process was repeated by superimposing more drops on the original spot. The TLC plates were developed with a

CH2Cl2/ MeOH solvent system. The developed plates were visualized under UV light (short and long wave length) and the components showing UV absorbance and florescence were marked. Afterwards the TLC plates were sprayed with spray reagents for the further localization of the interesting zones. Following spray reagents were used for this purpose. Anisaldehyde/sulphuric acid (Table 2.53). Ehrlich’s reagent to determine the presence of indoles and other nitrogen containing compounds. (Table 2.54). Ninhydrin (Table 2.55). Chlorine/ o-dianisidin for detection of peptides (Table 2.56). Palladium (II)-chloride for the detection of sulfur compounds. (Table 2.57). NaOH for the detection of peri-hydroxy quinines (Table 2.58).

Peptide Test

Peptide test was performed to detect the presence of peptides in a crude extract. A developed TLC plate having the crude extract was lightly sprayed with water and was placed in chlorine atmosphere (in a TLC chamber having KMNO4 + few drops of HCL in a separate vial) for 30 min. Later the TLC plate was aerated until the smell of chlorine was completely finished and the TLC plate was sprayed with spray reagent (Peptide reagent). The presence of peptides was indicated by the appearance of blue spots on TLC.

Materials and Methods

High Performance Liquid Chromatography (HPLC)/ ESI-MS

Sample Preparation

Sample preparation is a topic of high importance when an LC/MS/MS method is developed to assay biological samples. In addition, the method’s performance should remain reasonably consistent over time. The results should be relatively free from systematic error, any relative error should be characterized and consistent and meet acceptability guidelines for the method. Therefore, sample preparation is used to ensure that a method maintains certain basic elements of ruggedness and consistency that are expected in any assay. Generally a concentration of 1 to 5000 ng/ml is needed. The solvent used was methanol.

Chromatographic and Mass Spectrometry Conditions

For the chromatographic separation, the column used was RP-C12- column from Phenomenex with 150 mm length. RP-C12 possesses a slighter number (25%) of free Silane groups and results in better divided characteristics, especially for basic and tailing compounds. As a mobile phase, a stepwise/linear binary methanol/water-gradient with 0.05% formic acid (increase the sharpness and quality of the signal peaks) was used. At room temperature and at time zero a mixture of 90% water and 10% methanol was flushed through the column and increase to 100% methanol in 20 min. At this concentration the gradient was maintained for 10 min and set down subsequently during 2 min to 10% Methanol again and held therefore the final 8 min of the run (Figure 2.1). The optimal flow rate was 300µl/min and a volume of 5 µl was injected on to the analytical column which is connected directly to the UV/VIS-DAD detector with a detection wavelength was in the range of 200 nm to 800 nm.

The protective method ESI (Electrospray Ionisation) was applied in positive and in negative polarity at the electrospray voltage of 4.50 kV and the dwell time was 50 ms in the full scan and 200ms in SIM (selected ion monitoring) scan with a 3ms pause between scans. The capillary temperature was adjusted at 220 °C. The source was operated in both ions mode using an 80 psi nitrogen sheath gas. The instrument was scanned from 100 to 2000 amu. Due to fluctuations in mass assignment, a single ion is allowed to have a width

Materials and Methods

of ± 0.5 m/z. Ions also have a width along the time axis corresponding to chromatographic peak width. In the most cases the quasi monomer ion is given as [M+H]+, [M+Na]+, [M-H]- as well as dimmer ion [2M+H]+, [2M+Na]+ and [2M-H]-, [2M+Na-2H]- etc.

100

40 MeOH/H2O [%] MeOH/H2O 30

0 0 5 10 15 20 25 30 35 40 t [min] Figure 2.1: Applied Methanol/Water gradient Sometime the signal intensity of the dimmer ion is to high compare to those of the monomer ion. To enable this, several investigation were done relating to the ion source collision-induced dissociation (ISCID), which can allow the dimmer to decompose back into the monomer, at the end it was found that the collision energy around ISCID: 10V was favoured because of the stability of different molecules.

HPLC-MS Specifications: Masspectrometer: Finnigan LCQ; UV/VIS-Detektor: Finnigan Surveyor PDA Detector (Thermo Electron Corporation); HPLC-pump: Rheos 4000 (Flux Instrument); Deaerator: ERC-3415α (Flux Instruments); Autosampler: Jasco 851-AS Intelligent Sampler (Jasco); Control software HPLC: Janeiro (Flux Instruments); File system: Xcalibur (Finnigan); Databank software: MS-Manager (ACDLabs); column: EC 125/2 Nucleosil 100-5 C18 (Macherey-Nagel), Synergi 4μ MAX-RP 80A, 150×2.00 mm 4μ micron (Phenomenex); Solvent: Methanol LiChrosolv hypergrade for Liquid chromography (Merck). Programm: start 10% Methanol to 100% Methanol in 20 min, 10 min 100 % Methanol, from 100 % Methanol to 10 % Methanol in 2 min.; flow rate: 300 μl/min.

FERMENTATION The cultivation and scale-up steps were carried out only after the prescreening. As most strains are producing metabolite concentrations of only 0.1–1 mg/l, fermentation in at

Materials and Methods

least 20-50 liters scale is necessary to get an adequate amount of the product. There were two possibilities available for culturing the selected strains, the fermentation in shaking flasks on a linear shaker and fermentation in lab fermentors of 20 and 50 liter size.

Cultivation as Shaking Cultures

The 20 L of GYM medium (Table 2.5) was prepared in large containers and the pH was adjusted to 7.8 with 2N HCl and 2N NaOH. The medium broth was dispensed in 80 1L Erlenmeyer flasks with inflections, each containing 250 ml of the GYM medium (Table 2.5). The flasks were covered with cotton plugs and were autoclaved (Autoclave: Albert Dargatz Autoclave, volume 119 l, working temperature 121 °C, working pressure 1.2 kg/cm2). Each of the flasks was inoculated separately by an agar plug cut from a well grown plate of the selected Streptomyces strain. The fermentation was carried out at 95 rpm on the linear shaker (Infors AG (CH 4103 Einbach) type ITE, Laboratory shaker: IKA-shaker type S50 (max. 6000 Upm) for 7 days at 28o C. It is worth mentioning here that, most of the coloured compounds (e.g. quinones), are produced better in shaker culture than in a jar fermenter.

Fermentation in 20 And 50 L Fermenters

20 L fermenter (Biostat E, Fa. Meredos GmbH, Göttingen) consisting of culture container, magnet-coupled propeller stirrer, cooler with thermostat, control unit with pH and antifoam regulation, the 50 L fermenter (Biostat U) consisted of a 70 L metallic container (50 L working volume), propeller stirrer, and culture container covered with thermostat for autoclaving, cooling and thermostating (Braun Melsungen, Germany) were used for the fermentation of selected strains. This is a two-step process the preparation of precultures and the actual fermentation. The precultures were prepared by inoculating the strains from well grown culture plates into 1 litre Erlenmeyer flasks each having 250 ml of GYM medium. The flasks were incubated at 28 oC on a linear shaker for 4 days. Preculture with the ratio of 10% was used to inoculate the biosystem. The fermentors were filled with 20 or 50 litre water as the case may be along with appropriate quantity of

GYM medium (Table 2.5). The medium contents were mixed by stirring for some time

Materials and Methods

and the fermenter was closed with the metal lid. The inlet and outlet openings and tubes were also closed with stoppers and clamps. The pH electrode port was closed with the metal stopper. The fermenter was set to sterilization mode and the medium was autoclaved for 30 minutes at 121oC. After cooling of the system, the preculture of a selected strain with the ratio of 10% of the total volume was transferred to the fermenter under sterile conditions. Afterwards the air supply, stirring motor and water circulation pumps were switched on. The temperature was adjusted at 28o C and the pH was adjusted to 6.5+- 1.5. The acid 2N HCl (2.60), base 2N NaOH (2.58) and antifoam 1% Niax/ 70% ethanol (Niax PPG 2025; Union Carbide Belgium N. V. Zwiijndrecht) (Table 2.59) were filled in their respective jars and connected to the system. The pH electrode was sterilised with 70% ethanol and adjusted in the pH electrode port in the lid. The working of the system was monitored and the observations were recorded thrice a day for, Stirring rate, air supply, quantity of acid, base and antifoaming agent used, along with change in colour of the culture broth. The Fermentation was carried out for 5 days and after the strain has grown well the culture broth was harvested for extraction and further purifications of the bioactive metabolites.

FILTRATION

The culture broth with well grown cell mass and produced bioactive compounds was harvested in large containers from the shaking culture or fermentors as the case may be. The culture broth was filtered through filter press (Schenk Niro 212 B40). The culture broth was transferred to the port of filter press and mixed with celite (Celite France S. A., Rueil-Malmaison Cedex). The filters (Sterile filters: Midisart 2000, 0.2 μm, PTFE-Filter, Sartorius, Göttingen), soaked in water was adjusted in the separating chambers of the filter press and the pressure was applied. The filtration process resulted into the cell mass or mycelial cake and the filtrate or the liquid phase, which were further extracted separately.

EXTRACTION

The cell mass or the mycelial cake was extracted with ethyl acetate and acetone, the process of extraction continued until no further colour was obtained. One to two liters of

Materials and Methods

the solvent was mixed with the mycelial cake and stirred for half hour then the mixture was sonicated for further half hour (Ultraturrax: Janke & Munkel KG), the process was repeated three times. Extraction can be considered complete when little or no additional residue is obtained after concentrating the solvent. The storage of the resulting crude extracts at room temperature can lead to degradation of the compounds and lower overall yields. It is strongly recommended that the Solutions should therefore be evaporated as soon as possible, and the residues should be stored at coldest possible temperature in order to minimize the degradation of compounds. So the resulting crude extracts from the cell mass by the extraction with ethyl acetate and acetone were stored in deep freezing section (-20 oC) of the lab refrigerators.

The filtrate or liquid phase of the culture broth was extracted by adsorption on XAD resin in a large size glass column. Adsorption on XAD resin is another efficient extraction method for obtaining the crude extracts. For this purpose, the culture filtrate is passed at a suitable flow rate through a glass column containing XAD resin (XAD-2). Later the compounds are eluted from XAD usually with methanol or a methanol/water gradient. Extraction of the filtrate or liquid phase with XAD is more advisable than the commonly used ethyl acetate extraction in a separating funnel because it is considered as more economical and fast. Highly polar water-soluble compounds can also be obtained by this method as lipophilic interactions are possible. Over all good recovery rates can be achieved and it is easy to recover and purify the resin for further use. In contrast to solvents, the resin is not harmful to the bioactive compounds present in the filtrate. The isolation depends mainly on the polarity of the compounds of interest (which can be determined by thin layer chromatography with eluents of varying polarity). In this method the filtrate or liquid phase of a selected strain was passed through XAD column at the flow rate of 5-10 ml/min. The XAD resin was eluted with pure methanol, for the filtrate volume of 20L about 5-10L of the methanol and for the filtrate volume of 50 L 15-20 L of methanol was used. The methanol having bioactive metabolites was evaporated on the rotavapour (Rotavapor R152) and the resulting few grams of residue (the crude extract) was stored in the deep freezing section of the lab refrigerators for further purifications of its different components.

Materials and Methods

PURIFICATION OF THE BIOACTIVE COMPONENTS

There are a number of preliminary systems, which are commonly suitable for the separation and purification of most of the metabolites, the most famous among them includes: Column Chromatography, Preparative TLC, and Gel Exclusion Chromatography.

Column Chromatography

Adsorption chromatography in biomolecular applications usually consists of a solid stationary phase and a liquid mobile phase. The stationary phase is confined to a glass tube and the mobile phase is allowed to flow through the solid adsorbent. A small amount of the sample to be analyzed is layered on top of the column. The sample mixture enters the column of adsorbing material and is distributed between the mobile phase and stationary phase. The various components in the sample have different affinities for the two phases and move through the column at different rates. Collection of the liquid phase emerging from the column yields separate fractions containing the individual components in the sample. Specific terminology is used to describe various aspects of column chromatography. When the actual adsorbing material is made into the column, it is said to be packing of the column, application of the sample to the top of the column is called loading of column and movement of the solvent through the loaded column is called developing or eluting the column. The bed volume is the total volume of solvent and adsorbing material taken up by the column. The elusion volume is the amount of solvent required to remove a particular solute from the column. 20 to 50 cm long glass columns with inside diameter between 0.5 to5 cm were used. MN silica gel 60: 0.05- 0.2 mm, 70-270 mesh (Macherey-Nagel & Co); silica gel for flash chromatography: 30-60 μm (J. T. Baker); Flash chromatography of the crude extracts on silica gel was done, in this method an appropriate quantity of the crude extract was loaded over the silica gel column and a stepwise gradient of dichloromethane/methanol or ethyl acetate/cyclohexane was applied. This system separated the fractions depending on their polarity. The fractions were collected manually and sometime by an autocollector.

Materials and Methods

Disadvantage of this method is the contact of metabolite with silica gel, as it may rearrange, oxidise, cleave or even destroy metabolites.

Preparative TLC

Preparative TLC was used for the purification of the compounds present in mixtures of two to three components. In this method 55 g Silica gel P/UV254 (Macherey-Nagel & Co.) was added to 120 ml of demineralised water with continuous stirring for 15 minutes. 60 ml of the homogenous suspension was poured on a horizontal held (20 × 20 cm) glass plate and the unfilled spaces were covered by distributing the suspension. The plates were air dried for 24 hours and activated by heating for 3 hours at 130 °C. An appropriate quantity of the sample was loaded on the PTLC and the plates were developed in dichloromethane/methanol solvent system. The multiple elutions of the plates were done sometime in order to obtain the maximum separation of the components of a mixture. The part of the PTLC plate having a fraction was scraped and dissolved in solvent, after filtration the solvent was evaporated on a rotary evaporator to get the purified fraction.

Gel Exclusion Chromatography

The method of gel exclusion chromatography exploits the physical property of molecular size to achieve separation. The stationary phase consists of inert particles that contain small pores of a controlled size. Microscopic examination of a particle reveals an interior resembling a sponge. A solution containing solutes of various molecular sizes is allowed to pass through the column under the influence of continuous solvent flow. Solute molecules that are larger than the pores cannot enter the interior of the gel beads, so they are limited to the space between the beads. The volume of the column that is accessible to very large molecules is, therefore, greatly reduced. As a result they are not slowed in their progress through the column and elute rapidly in a single zone. Small molecules that are capable of diffusing in and out of the beads have a much larger volume available to them. Therefore they are delayed in their journey through the column bed. Molecules of intermediate size migrate through the column at a rate somewhere between those for large and small molecules. Therefore the order of elution of the various solute molecules is directly related to their molecular dimensions.

Materials and Methods

In order to achieve the maximum purity of the separated fractions from silica gel column chromatography and PTLC, most of the compounds were finally purified by gel exclusion chromatography using Sephadex LH-20. Sephadex does not have the disadvantages like rearranging, oxidizing, cleaving or degrading the metabolites and the recovery rate for the compounds is also high. The afforded fractions were then analyzed by mass spectrometery and NMR spectroscopy along with search in literature data for their identification and structural elucidation.

IDENTIFICATION AND STRUCTURE ELUCIDATION

The purified fractions were analyzed by mass spectrometry (MS) and NMR spectroscopy for their identification and structure elucidation. The data obtained was compared with the reference data present in databases like Dictionary of Natural Products (DNP), Anti Base (a database of the microbial metabolites) and chemical abstracts.

Mass Spectrometry (MS)

Mass Spectrometry is a powerful technique for identification and studying molecular structure of the unknown metabolites. Mass spectrometry is essentially a technique for "weighing" molecules. Obviously, this is not done with a conventional balance or scale. Instead, mass spectrometry is based upon the motion of a charged particle, called an ion, in an electric or magnetic field. The mass to charge ratio (m/z) of the ion affects this motion. Since the charge of an electron is known, the mass to charge ratio a measurement of an ion's mass. A variety of ionization techniques are used for mass spectrometry. Most ionization techniques excite the neutral analyte molecule which then ejects an electron to form a radical cation (M+). Other ionization techniques involve ion molecule reactions that produce adduct ions (MH+). The most important considerations are the physical state of the analyte and the ionization energy. Electron impact (EI) and chemical ionization (CI) are only suitable for gas phase ionization. Fast atom bombardment (FAB), secondary ion mass spectrometry, electrospray ionization (ESI), and matrix assisted laser desorption (MALDI) are used to ionize condensed phase samples. The ionization energy is significant because it controls the amount of fragmentation observed in the mass

Materials and Methods

spectrum. Although this fragmentation complicates the mass spectrum, it provides structural information for the identification of unknown compounds.

Mass spectra: The purified fractions were analyzed by EI MS at 70 eV with Varian MAT 731, Varian 311A, AMD-402, ESI MS with Quattro Triple Quadruple mass spectrometer Finigan MAT-Incos 50, ESI-MS LCQ (Finnigan) and ESI-HRMS with perflurokerosine as standard. Electrospray Ionisation (ESI) was generally applied to the analysis of polar molecules. In this method, the samples were dissolved in a polar, volatile solvent and pumped through a narrow, stainless steel capillary (75 - 150 micrometers i.d.) at a flow rate of between 1 µL/min and 1 mL/min. A high voltage of 3 or 4 kV was applied to the tip of the capillary, situated within the ionization source of the mass spectrometer, as a consequence of this strong electric field, the sample emerging from the tip is dispersed into an aerosol of highly charged droplets, a process that is aided by a co-axially introduced nebulising gas flowing around the outside of the capillary. This gas, usually nitrogen, helps to direct the spray emerging from the capillary tip towards the mass spectrometer. The charged droplets diminish in size by solvent evaporation, assisted by a warm flow of nitrogen known as the drying gas, which passes across the front of the ionisation source. Eventually charged sample ions, free from solvent, are released from the droplets, some of which pass through a sampling cone or orifice into an intermediate vacuum region, and from there through a small aperture into the analyzer of the mass spectrometer, which is held under high vacuum. In positive ionization mode, a trace of formic acid was often added to aid protonation of the sample molecules; in negative ionisation mode a trace of ammonia solution or a volatile amine was added to aid deprotonation of the sample molecules. The peptides components were usually analyzed under positive ionization conditions.

Nuclear Magnetic Resonance (NMR) Spectroscopy

Nuclear magnetic resonance, or NMR as it is abbreviated, is a phenomenon which occurs when the nuclei of certain atoms are immersed in a static magnetic field and exposed to a second oscillating magnetic field. Some nuclei experience this phenomenon, and others do not, dependent upon whether they possess a property called spin. Spectroscopy is the

Materials and Methods

study of the interaction of electromagnetic radiation with matter. Nuclear magnetic resonance spectroscopy is the use of the NMR phenomenon to study physical, chemical, and biological properties of matter. As a consequence, NMR spectroscopy finds applications in several areas of science. NMR spectroscopy is used to study chemical structure using simple one-dimensional techniques. Two-dimensional techniques are used to determine the structure of more complicated molecules. The versatility of NMR makes it pervasive in structural elucidation of the unknown metabolites. Both 1H NMR spectra and 13C NMR spectra were measured: 1H NMR spectra were measured by: Varian Unity 300 (300 MHz), Bruker AMX 300 (300 MHz), Varian Inova 500 (499.8 MHz). Coupling constants (J) in Hz. Abbreviations: s = singlet, d = doublet, dd = doublet doublet, t = triplet, q = quartet, m = multiplet, br = broad. 13C NMR spectra were measured by: Varian Unity 300 (75.5 MHz), Varian Inova 500 (125.7 MHz). Chemical shifts were measured relative to tetramethylsilane as internal standard. Abbreviations: APT (Attached Proton Test): CH/CH3 up and Cq/CH2 down.

CULTURE OPTIMIZATION

Streptomyces have the ability to grow on variety of media and under different culture conditions; however it is usually advantageous to find out the optimal medium, temperature, pH and aeration of the interesting strains for large scale cultivation. Two strains were selected in this experiment in order to find out the appropriate medium and optimal culture conditions for the growth and maximum production of the metabolites produced by them. Following four sets of culture conditions were applied.

CC1: pH 6.5, incubation temperature 28oC, shaking at 95 rpm, for 7 days.

CC2: pH 6.5, incubation temperature 35oC, shaking at 110 rpm, for 7 days.

CC3: pH 7.8, incubation temperature 28oC, shaking at 95 rpm, for 7 days.

CC4: pH 7.8, incubation temperature 35oC, shaking at 110 rpm, for 7 days.

The six different media used for this purpose are mentioned in Tables 2.31-2.37. Three litres of each of the six media were prepared and were dispensed into twelve one litre flasks (250 ml broth in each flask). The flasks were arranged into groups according to the

Materials and Methods

proposed culture conditions, so that under each culture condition there were three replicates R1, R2 and R3 (three one litre flasks containing 250 ml media each), pH was adjusted in each of the flask and the flasks were sterilized by autoclaving and were inoculated by test strains from well grown plates. The inoculated flasks were incubated on linear shakers under the proposed culture conditions CC1, CC2, CC3 and CC4. The strains were harvested after seven days, the culture broth in each of the flask was freeze dried and was extracted individually three times with ethyl acetate. After evaporating the ethyl acetate on a rotary evaporator the crude extracts were obtained. The crude extracts were weighed to determine the quantity of bioactive metabolites produced/250 ml of culture broth under different media compositions and different sets of cultural conditions (mean weights of the crude extract/250ml of culture broth for three replicates were taken). Later the crude extracts were tested for antimicrobial activity against a set of 3 test strains, including Bacillus subtilus, Staphylococcus aureus, Streptomyces viridochromogens TU57, to find out the relative concentration of the active components in a unit weight of the crude extract. The antimicrobial activity was determined by disc diffusion method, 40 µg of the extract was loaded on each disc and the discs were transferred aseptically to the surface of the agar plates containing lawn of the test organisms, the diameter of the zones of inhibition were measured and the means of three replicates were taken. The results obtained were analysed statistically applying analysis of variance (ANOVA) test (Duncan’s multiple range test) using SPSS software version 10 at (P=0.001).

MUTATIONAL ANALYSIS

For mutational analysis two chemical mutagens, ethidium bromide, N- methyl N- nitro- N- nitrosoguanidine (MNNG or NTG) and two physical mutagens (gamma radiations and UV at 254 nm) were selected. In case of ethidium bromide, MNNG and UV treatments spore mass was used, however for gamma irradiation mycelium of the wild strain was used. The mutants were selected on the basis of resistance against chloramphenicol; while the mutant selection in treatments with gamma radiations was random i.e. the derivatives which exhibited enhanced activity were selected randomly.

Materials and Methods

Determination of the Antibiotics Sensitivity Pattern of the Parental Strain

The antibiotic sensitivity pattern of the selected wild Streptomyces strains used in mutational analysis was determined by the standard Kerby-bauer disc diffusion method (Bauer et al., 1966), against the antibiotics: Novobiocin, Oxytetracycline, Chloramphenicol, Gentamycin Ampicilin, Penicillin, Carbenicillin and Erythromycin (Table 2.61). On the basis of sensitivity pattern obtained, the antibiotic chloramphenicol was selected as a selection marker for the isolation of mutants or derivatives of the wild strain.

Determination of MTC (Maximum Tolerable Concentration) of Chloramphenicol for the Parental Strain

The wild Streptomyces strain was inoculated on GYM agar plates containing twelve different concentrations of chloramphenicol (1μg/ml, 2μg/ml, 3μg/ml, 4μg/ml, 5μg/ml, 6μg/ml, 7μg/ml, 8μg/ml, 9μg/ml, 10μg/ml, 15μg/ml, and 20μg/ml) and was incubated at 28°C for 10 days. The growth of the wild strain was observed at different intervals and was recorded. The maximum concentration that allowed the growth of the organism was considered to be the MTC (Maximum Tolerable Concentration) of Chloramphenicol for the wild strain.

Preparation of Spore Suspensions

Spores of the Streptomyces usually arise as chains in the aerial mycelium; however individual spores can readily be obtained by suspending and vortexing the spores in water, a wetting agent such as 0.1 % Tween 80 or 0.001 % Triton X100 is added to the suspension. The resulting suspensions can be used for many purposes, such as inoculating liquid medium to produce mycelium for isolating plasmid or chromosomal DNA, RNA or enzymes, or for preparing protoplasts; for the isolation of mutants etc. Spores suspensions in 20% glycerol, frozen at -20°C, usually remain viable for years, even if they are repeatedly thawed and re-frozen for sampling purposes. Spore suspensions frozen in water usually lose about 50% of their viability on freezing and thawing. Autoclaved water or 0.9% saline, 5-10 ml was added to the plate containing well grown wild strain with spores. The surface of the culture was scraped with a hard, sterilized

Materials and Methods

inoculating loop, with gentle pressure and then gradually more vigorously, to suspend the spores. The crude suspension containing the spores was transferred to a sterile test tube, using a sterile syringe. The suspension was agitated on a vortex for about one minute to break up the spore chains. The number of spores/ml was adjusted by measuring the optical density of the spore suspension at 600nm (Cecil-CE 7200/7000 series spectrophotometer). The optical density of the spore suspensions was adjusted to 1 using autoclaved distilled water/ normal saline. At O.D =1 the number of spores in the suspension is about 106 spores/ml (Kieser et al., 2000).

Mutagenesis by Ethidium Bromide

Clean, pre-autoclaved appendorfs were oven dried before use and 500μl spore suspension of the wild strain was aseptically transferred to separate appendorfs. Appropriate quantity of stock solution of ethidium bromide (Table 2.63) was added to each appendorf to get the four different working concentrations (10μg/ml, 20μg/ml, 50μg/ml and100μg/ml) of ethidium bromide. Later the suspension volume was made up to 1000μl with the sterile normal saline. The spore suspensions were incubated for approximately 1 hour at 28°C. After the incubation the spore suspensions were thoroughly mixed by vortexing for about one minute and were centrifuged at 12,000 rpm for 5 minutes and the supernatant was discarded. The spore pellet was washed thrice with 0.5 M EDTA to remove traces of ethidium bromide from the spore mass, and the spores were resuspended in 0.9% saline solution. A set of control without treatment with ethidium bromide was run along with the experimental spore suspensions.

Mutagenesis by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)

Clean, pre-autoclaved appendorfs were oven dried before use and 500μl spore suspension of the wild strain was aseptically transferred to each separate appendorf and was centrifuged at 12,000 rpm for 2 minutes. Then the spores were resuspended in the TM buffer (Table 2.65) 800μl and were vortexed for 2 minutes to resuspend the spore pellet. Appropriate volume of the stock solution of MNNG (Table 2.64) was added to each appendorf to get the four different working concentrations (250μg/ml, 500μg/ml, 750μg/ml and 100010μg/ml) of MNNG and was immediately mixed on a vortex for

Materials and Methods

about 2 minutes. A set of control without treatment of MNNG was run, along the experimental spore suspensions. The spore suspensions mixed with MNNG were incubated at 30°C for 2 hours. After the incubation the spore suspensions were thoroughly mixed by vortexing vigorously for about one minute and were centrifuged at 12,000 rpm for 5 minutes and the supernatant was discarded. The spore pellet was washed thrice with autoclaved distilled water to remove traces of MNNG from the spore suspensions and was resuspended in normal saline.

Mutagenesis by Ultraviolet Light (UV at 254 nm)

The UV lamp was switched on for at least 15 min before use to allow it to reach the maximum emission. The 10 ml of spore suspensions (approximately containing 106 spores/ml) of individual strains were placed separately in sterilized glass Petri dishes. The spore suspensions were irradiated under the short wavelength (254nm) UV, with constant agitation on a flatbed shaker for the time intervals 15, 30, 45 and 60 minutes. The UV treatments were carried out at sufficiently low light levels and the treated spores were immediately incubated in dark to avoid photo reactivation (Held and Kutzner, 1991).

Selection and Maintenance of the Chloramphenicol Resistant Mutants

After the mutagenic treatments with ethidium bromide, N-methyl-N'-nitro-N- nitrosoguanidine (MNNG) and UV at 254nm, 50μl of spores suspension from each of the individual treatment was spreaded on the GYM agar (Table 2.6) plates containing chloramphenicol as the selective marker, the plates were incubated for 5-10 days at 28°C. The individual colonies grown were considered to be chloramphenicol resistant mutants, the colonies were selected and purified on GYM agar (Table 2.6). The selected mutants were screened for change in the antimicrobial activity (increase, decrease and even loss in activity) against Gram positive bacteria Bacillus subtilus, by the effect of mutagenesis. The selected mutants were stored as agar slants at low temperature (-20°C).

Mutagenesis with Gamma Radiations

The effect of Gamma irradiation was determined on the antimicrobial activity of the selected wild strain. The strain was grown on small scale as shaking culture in GYM

Materials and Methods

medium (Table 2.5) and the culture broth was centrifuged for obtaining the cell mass. The cell mass was suspended in demineralised autoclaved water and 0.9 % saline in 1.5 ml microtubes. The microtubes were labelled and separated into groups according to the doses of radiations. The microtubes containing the cell mass were subjected to different gamma irradiation doses 0, 0.5, 1, 2, 5, 10 and 20 Gy using 60Co as a source.

After exposure to radiations the colonies from each sample in the microtubes were streaked on GYM agar plates and the plates were incubated at 28oC for 7 days. After incubation 15 colonies were selected randomly from each plate as the suspected variants. Later each of the selected colonies was grown separately as shaking culture in a vial containing 10 ml GYM broth on an orbital shaker at 28oC for 11 days. The antimicrobial activity was determined three times at different intervals i.e. 5th day, 8th day and 11th day. 1 ml of the culture broth was harvested from the vial each time and was centrifuged for getting supernatant, 50 µL of the supernatant was loaded in each well and the zone of inhibition was measured in mm. The variants or mutants were selected on the basis of change (significant decrease or significant increase) in antimicrobial activity with reference to the activity of the control. Afterward the selected variants were subjected to metabolite pattern analysis, in this method the culture broth of each variant was freeze dried (Christ Alpha 2-4 LD) and was extracted with ethyl acetate. The crude extracts were obtained after evaporating the solvent and were spotted on the TLC plates. The TLC plates were developed in a dichloromethane/methanol solvent system and were sprayed with spraying reagents, anisaldehyde/H2SO4 and Eherlich’s reagent. The metabolite patterns of the variants were compared with that of the control.

STATISTICAL ANALYSIS

Where ever applicable the results obtained were analyzed statistically following Steel and Torrie (1981). The data were presented in terms of arithmetic averages of at least three replicates unless otherwise indicated and the error bars indicated the standard deviations. The analyses were carried out using SPSS software, version 10 (SPSS Inc., Chicago, USA).

Isolation and characterization of indigenous bioactive streptomycetes CHAPTER 3

ISOLATION AND CHARACTERIZATION OF INDIGENOUS BIOACTIVE STREPTOMYCETES

In natural soil habitats, streptomycetes are common and are usually a major component of the total actinomycetes population (Hayakawa, 2008). For the selective isolation of microorganisms the following basic stages have been recommended (Williams and Welington, 1982; Williams et al., 1984). 1- choice of the material containing microbes, 2- enrichment and pretreatment of the sample, 3- use of selective media as well as incubation conditions, 4- colony selection and purification. There is no single selective procedure for the isolation of streptomycetes from soil, good success is obtained using a variety of strategies. The strategies are designed to select against fast growing bacteria and fungi. Incorporation of antifungal antibiotics such as cycloheximide and nystatin effectively eliminate fungi. Thorough drying of the soil sample at ambient or elevated temperature kills a great many of the resident bacteria, particularly Gram-negative bacteria, while the more desiccation resistant streptomycetes survive (Tamura et al., 1997). Several physical barriers such as filtration, centrifugation and chemical treatment methods are also employed for the selective isolation of streptomycetes from the soil samples (Nonomura and Hayakawa, 1988). The members of streptomycetes are chemoorganotrophic, non fastidious microorganisms, so very simple “synthetic media” containing an organic carbon source (glucose, starch, + - glycerol, or lactate), an inorganic nitrogen source (NH4 , NO3 ), and some mineral salts, can be used for their cultivation. However to obtain faster growth than that achieved on such substrates, “complex media” containing oat meal, yeast extract and malt extract are generally used. The isolated strains can be stored on long term and short term basis, for short term storage, agar slope cultures may be stored at 4˚C for few months, spore suspensions can be mixed with soft agar water and glycerol (10% v/v) and stored at - 20˚C (Kutzner, 1972, Wellington and Williams, 1978). For long term storage, preservation as soil cultures (Pridham et al., 1973), lyophilization (Hoopwood and Ferguson, 1969), and storage in the vapor phase of liquid nitrogen are most generally employed methods. The criteria employed for the characterization of an unknown 64

Isolation and characterization of indigenous bioactive streptomycetes Streptomyces isolate includes: biochemistry, macroscopic appearance, morphology, physiology, patterns of enzymes and proteins, serology and molecular genetics. Morphologic and physiological characterization is considered sufficient to place an organism into a particular family of the actinomycets.

For the work described here, the soil samples were collected from different localities, including botanical garden and adjacent rose cultivated fields of University of the Punjab, Lahore, saline agricultural farm lands in district Jhang of the province Punjab and Northern areas of Pakistan. On the whole 30 soil samples were collected, among them 15 samples came from saline agricultural farm lands, including corn fields, cotton fields and sugar cane fields (Table 3.1). Ten of the soil samples were collected from botanical garden and rose fields, while five samples belong to the northern areas of Pakistan. The soil samples were collected batch wise at different times and different seasons in sterile plastic bags. Later the soil samples were treated physically and chemically, for the selective isolation of streptomycetes. The selective media used for this purpose is mentioned in Table 2.1-2.4. A preliminary antimicrobial activity test was performed at this stage and only the bioactive strains were selected for further work. In actual 110 bioactive Streptomyces strains were selected from the 30 soil samples (Table 3.1-3.6). Figure 3.1 summarizes the strategy adopted for the isolation, purification and characterization of these indigenous bioactive Streptomyces strains.

The soil samples were serially diluted and the dilutions 10-3 and 10-4 were plated in duplicates on selective media (Table 2.1-2.4), supplemented with nystatin. The culture plates were incubated at 28˚C for three to four weeks. The Streptomyces colonies were selected and the strains were purified by repeated sub culturing. From all the 30 soil samples of different origin (Table 3.1) 110 bioactive Streptomyces isolates were selected on the basis of preliminary antimicrobial activity (Table 3.2-3.6). The whole Streptomyces isolates collection was divided into five major groups on the basis of origin of the strains from different soils. Group 1 includes 24 strains (Table 3.2) that were isolated from the soil of botanical garden and adjacent rose fields. The actual number of the Streptomyces strains obtained from these 10 soil samples was high, but in preliminary

Isolation and characterization of indigenous bioactive streptomycetes

30 soil samples

Agricultural farm lands

Sample treatment

Physical treatment Chemical treatment

High Enrichment temperature, with CaCO 3 centrifugation, and sodium and filteration hypochlorite

1) Serial dilutions, plating on selective media and incubation at 28o C for 7-21 days

2) Selection of actinomycetes colonies and purification of the cultures by repeated sub culturing 3) Antimicrobial activity test and final selection of the strains (110 bioactive strains)

Culture preservation Taxonomic studies

Morphological and Biochemical and Ribotyping(16S rRNA microscopic analysis physiological testing gene sequencing)

Figure 3.1 working up scheme for the isolation and characterization of indigenous Strptomyces strains from soil.

Isolation and characterization of indigenous bioactive streptomycetes

Table 3.1 Soil samples collected for the isolation of indigenous streptomycetes

Soil Sample Wt/depth of Nature of the Locality No. the sample sample

1 4 gm, 10 cm Organic matter rich soil Wild plants growing area, Botanical Garden 2 4 gm, 10 cm Organic matter rich soil Grassy lawn in Botanical Garden 3 4 gm, 10 cm Saline soil Banks of a pond in Botanical Garden 4 4 gm, 10 cm Dry soil Ploughed field in Botanical Garden 5 4 gm, 10 cm Organic matter rich soil Wheat field in Botanical Garden 6 3 gm, 10cm Organic matter rich soil Rhizosphere of rose, Rose Fields 7 3 gm, 10 cm Organic matter rich soil Rhizosphere of rose, Rose Fields 8 3 gm, 10 cm Moist soil Rose Field, University campus 9 3 gm, 10 cm Dry and hard soil Rose Field, University campus 10 3 gm, 10 cm Dry and hard soil Rose Field, University campus 11 4 gm, surface Moist, saline soil Corn Field, salt affected farmland, (Jhang) 12 4 gm, surface Moist, saline soil Corn Field, salt affected farmland, (Jhang) 13 4 gm, surface Dry, saline soil Corn Field, salt affected farmland, (Jhang) 14 4 gm, surface Dry, saline soil Corn Field, salt affected farmland, (Jhang) 15 4 gm, surface Dry, saline soil Corn Field, salt affected farmland, (Jhang) 16 4 gm, surface Dry, saline soil Cotton Field, salt affected farmland,(Jhang) 17 4 gm, surface Dry, saline soil Cotton Field, salt affected farmland,(Jhang) 18 4 gm, surface Dry, saline soil Cotton Field, salt affected farmland,(Jhang) 19 4 gm, surface Dry, saline soil Cotton Field, salt affected farmland,(Jhang) 20 4 gm, surface Dry, saline soil Cotton Field, salt affected farmland,(Jhang) 21 4 gm, surface Moist saline soil Sugar cane field, (Jhang) 22 4 gm, surface Moist saline soil Sugar cane field, (Jhang) 23 4 gm, surface Moist saline soil Sugar cane field, (Jhang) 24 4 gm, surface Moist saline soil Sugar cane field, (Jhang) 25 4 gm, surface Moist saline soil Sugar cane field, (Jhang) 26 4 gm, 10 cm Organic matter rich soil Ayubia Hills, Northern areas of Pakistan 27 4 gm, 10 cm Organic matter rich soil Ayubia Hills, Northern areas of Pakistan 28 4 gm, 10 cm Organic matter rich soil Nathia Galli, Northern areas of Pakistan 29 4 gm, 10 cm Organic matter rich soil Bara Galli, Northern areas of Pakistan 30 4 gm, 10 cm Organic matter rich soil Bara Galli, Northern areas of Pakistan

Isolation and characterization of indigenous bioactive streptomycetes antimicrobial activity test only 24 were found to be biologically active, among the strains of group 1, the isolates RSF17, RSF18, RSF23 and BG5 exhibited potent activity against Gram positive and Gram negative test organisms (Table 3.2) and were finally selected for further characterization and antibiotic screening.

Group 2 includes 18 strains (Table 3.3) isolated from saline soil of a corn field, among these strains the isolates, CRF1, CRF2, CRF11, CRF14 and CRF17 exhibited most prominent activities and were selected for further analysis. Group 3 includes 27 Streptomyces strains isolated from saline soil of a cotton field (Table 3.4), from this group the isolates, CTF9, CTF13, CTF14, CTF15, CTF20, CTF23, CTF24 and CTF25, were selected finally for characterization and antibiotic screening. Group 4 includes 28 strains (Table 3.5), among them three representative strains including SCF18, SCF25 and SCF31 were selected for further studies. Group 5 includes 13 strains isolated from the soil of northern areas (forest soil) (Table 3.6), among them isolates ST4, ST5, ST18, ST34, ST71 were active against both Gram positive and Gram negative test bacteria, while rest of the strains showed activity either against Gram positive or Gram negative test bacteria.

The selected strains were cultivated on GYM medium (Table 2.5-2.6) and preserved as glycerol stocks or in the vapor phase of liquid nitrogen for further analysis. The strains were characterized morphologically, biochemically and physiologically (Table 3.7-3.10). In morphological characterization, the strains RSF17, RSF18 (Fig 3.4 A) and RSF23 exhibited moderate to well grown substrate mycelia that vary in color from yellow to brown as in case of strain RSF23. The color of aerial mycelium in case of strains RSF17 and RSF18 is white that gradually turns to gray at the later stages of life cycle however the strain RSF23 produces only gray color aerial mycelium. The soluble pigments or diffused pigments in the cultures of strains RSF17, RSF18 and RSF23 were not observed, moreover these strains produces hard/embedded and round colonies with a diameter of 5 to 6 millimeter (Table 3.7). The strain BG5 produces pink to reddish substrate mycelium and off white to light gray aerial mycelium, along with significantly visible soluble pigments that diffuses in the surrounding into the culture medium. The colonies of the

Isolation and characterization of indigenous bioactive streptomycetes strain BG5 are hard, well embedded into the agar medium with a diameter of 4 millimeter (Table 3.7). The strains CRF1, CRF2, CRF11, CRF14 (Fig 3.4 B) and CRF17 (Fig 3.4 C) isolated from the soil of corn field, produce yellow substrate mycelia that turns to brownish at the later stages of life cycle, the color of aerial mycelia in these strains is mostly white except the strains CRF2 and CRF17 where the color varies from off white to gray. These strains also produce soluble pigments into the surrounding medium which give an overall brownish look to the culture on GYM agar medium. The colonies are hard, embedded in the agar and vary in size from 5-9 millimeter in diameter (Table 3.7). The strains SCF18, SCF25, and SCF31 exhibited moderately grown substrate mycelia and off white to gray colored aerial mycelia, production of soluble pigments was observed in these strains along with hard embedded colonies of small size with a diameter of 4 millimeter (Table 3.7). The strains CTF9 (Fig 3.4 D), CTF13, CTF14, CTF15 (Fig 3.4 E), CTF20 (Fig 3.4 F), CTF23, CTF24 and CTF25 isolated from saline soil of cotton field, produce dark brown substrate mycelia and white to dark gray colored aerial mycelia as in case of strain CTF15. The soluble pigments were not observed in the cultures of these strains and colonies are round hard and of small size, usually between 4- 5 millimeter in diameter (Table 3.7). In microscopic analysis most of the strains exhibited typical filamentous structures (Fig 3.5 A-D) and the spores were observed in the form of chains or clusters (Fig 3.5 E, F).

For physiological characterization the selected strains were tested for the determination of optimal growth temperature, for the production of melanin, ability to utilize different carbon sources (eight different sugars), utilization of organic acids, utilization of oxalates, hydrolysis of urea and hemolysis. In determination of the growth temperature range, it was observed that all the strains grew at 28˚ C and 37˚ C (Table 3.8), however the growth at 28˚ C was more prominent and colonies were hard and compact as compared to the growth at 37˚ C. The strains RSF17, RSF18, RSF23 and BG5 also exhibited growth at 20˚ C however none of the strain was able to grow at 10˚ C similarly no growth was observed at higher temperatures i.e. at 45˚ C and 60˚ C (Table 3.9). The media used for melanin production and the basal medium for sugar utilization is mentioned in Table 2.11

Isolation and characterization of indigenous bioactive streptomycetes and 2.12. The tests were performed as agar slant cultures, in case of melanin production the cultures were regularly observed for the appearance of melanoid pigments between

Table 3.2 Group 1: Streptomyces strains isolated from the soil of rose field and botanical garden and selected on the basis of preliminary antimicrobial activity

S. Streptomyces Preliminary Antimicrobial Origin No. isolates Activity B.subtilus E.coli 1 RSF-3 ++ + Isolated from the soil 2 RSF-4 + - samples collected from 3 RSF-5 ++ + rose fields and botanical 4 RSF-9 + - garden. The soil was 5 RSF-11 + - organic matter rich. 6 RSF-12 + - These soil samples 7 RSF-14 + - 8 RSF-16 + - yielded a good number of 9 RSF-17 +++ ++ Streptomycetes and most 10 RSF-18 +++ ++ of the isolates were found 11 RSF-19 + + to be active against 12 RSF-21 + - indicator microorganisms 13 RSF-22 ++ + (Bacillus subtilus and E. 14 RSF-23 +++ + coli). 15 RSF-24 + - 16 RSF-25 + - 17 RSF-29 + - 18 RSF-30 + - 19 BG-2 + - 20 BG-5 ++ + 21 BG-13 + - 22 BG-14 + - 23 BG-32 + - 24 BG-33 + - Highlighted strains exhibited promising antimicrobial activity and were selected for further work

Isolation and characterization of indigenous bioactive streptomycetes

Table 3.3 Group 2: Streptomyces strains isolated from the soil of corn field (saline soil) and selected on the basis of preliminary antimicrobial activity

S. Streptomyces Preliminary Antimicrobial Origin No. isolates Activity B.subtilus E.coli 1 CRF-1 ++ - 2 CRF-2 ++ - Isolated from the soil 3 CRF-3 + - samples collected from 4 CRF-4 + - 5 CRF-5 + - corn fields. The soil was 6 CRF-6 + - saline. These soil 7 CRF-8 + - 8 CRF-9 + - samples also yielded a 9 CRF-10 + - good number of 10 CRF-11 +++ + 11 CRF-13 + - streptomycetes and most 12 CRF-14 +++ + of them are active against 13 CRF-15 + - indicator 14 CRF-17 +++ ++ 15 CRF-18 + - microorganisms. 16 CRF-19 + - 17 CRF-20 + - 18 CRF-22 + - Highlighted strains exhibited promising antimicrobial activity and were selected for further work

A B

Figure 3.2 Crowding of Streptomyces on actinomycetes isolation agar medium (AIA), A= 10-2 dilution, B= 10-3 dilution, Streptomyces colonies were selected from the crowding and were purified to obtain pure cultures

Isolation and characterization of indigenous bioactive streptomycetes

Table 3.4 Group 3: Streptomyces strains isolated from the soil of cotton field (saline soil) and selected on the basis of preliminary antimicrobial activity

S. Streptomyces Preliminary Antimicrobial Origin No. isolates Activity B.subtilus E.coli 1 CTF-1 + - Isolated from the soil 2 CTF-2 + - samples collected from 3 CTF-3 + - cotton fields. The soil 4 CTF-4 + - was saline and dry. 5 CTF-5 + - Again the number of 6 CTF-6 + - bioactive Streptomycetes 7 CTF-7 + + 8 CTF-9 +++ ++ isolated from these soil 9 CTF-10 +++ ++ samples was high 10 CTF-11 + - 11 CTF-13 ++ - 12 CTF-14 ++ - 13 CTF-15 +++ ++ 14 CTF-16 + - 15 CTF-17 + - 16 CTF-18 + - 17 CTF-19 + - 18 CTF-20 +++ ++ 19 CTF-21 + - 20 CTF-22 + - 21 CTF-23 +++ + 22 CTF-24 ++ + 23 CTF-25 +++ + 24 CTF-26 + - 25 CTF-27 + - 26 CTF-29 + - 27 CTF-30 + - Highlighted strains exhibited promising antimicrobial activity and were selected for further work

Isolation and characterization of indigenous bioactive streptomycetes

Table 3.5 Group 4: Streptomyces strains isolated from the soil of a sugar cane field (saline soil) and selected on the basis of preliminary antimicrobial activity

S. Streptomyces Preliminary Antimicrobial Origin No. isolates Activity B.subtilus E.coli 1 SCF-1 + - Isolated from the soil 2 SCF-2 + - samples collected from 3 SCF-3 + - sugar cane fields. The 4 SCF-4 + - soil was saline and moist. 5 SCF-5 + + Most of the isolates were 6 SCF-6 + + found to be active against 7 SCF-7 + - 8 SCF-8 + - indicator 9 SCF-9 + - microorganisms. 10 SCF-12 + + 11 SCF-13 + - 12 SCF-14 + - 13 SCF-15 + - 14 SCF-16 + - 15 SCF-17 ++ + 16 SCF-18 +++ ++ 17 SCF-19 ++ + 18 SCF-20 + - 19 SCF-24 + - 20 SCF-25 +++ + 21 SCF-26 ++ + 22 SCF-27 + + 23 SCF-28 + - 24 SCF-29 + - 25 SCF-30 + - 26 SCF-31 +++ - 27 SCF-33 + - 28 SCF-35 + - Highlighted strains exhibited promising antimicrobial activity and were selected for further work

Isolation and characterization of indigenous bioactive streptomycetes

Table 3.6 Group 5: Streptomyces strains isolated from the soil of northern areas of Pakistan (forest soil) and selected on the basis of preliminary antimicrobial activity

S. Streptomyces Preliminary Antimicrobial Origin No. isolates Activity B.subtilus E.coli 1 ST-4 + ++ Isolated from the soil 2 ST-5 + + samples collected from 3 ST-6 - + northern areas of 4 ST-18 ++ - Pakistan (Forest soil). 5 ST-19 +++ - The soil was organic 6 ST-34 ++ - matter rich and mixed 7 ST-47 - + 8 ST-48 - + with litter. These soil 9 ST-62 + - samples yielded 10 ST-71 + + relatively low number of 11 ST-82 - ++ bioactive Streptomyces 12 ST-83 - ++ 13 ST-90 - + Highlighted strains exhibited promising antimicrobial activity and were selected for further work

E. coli Bacillus subtilus

A B

Figure 3.3 Preliminary antimicrobial activities of the Streptomyces isolates determined by the solid media bioassay, the active isolates were selected for further work, while the inactive strains were rejected

Isolation and characterization of indigenous bioactive streptomycetes

Table: 3.7 Morphological characteristics of the selected Streptomyces strains after incubation at 28˚C for 7 days on GYM agar plates

Strain Growth pattern Color of Mycelium Soluble Colony characteristics s Pigments Substrate Ariel Consistency Size Shape Mycelium Mycelium mm

RSF 17 Moderate growth/partioned Brownish White-Gray - Hard/embedded 5 Round

RSF 18 Well grown/partioned Yellowish White - Hard/embedded 6 Round

RSF 23 Moderate growth/smooth Brownish Gray - Hard/embedded 5 Round

BG 5 Modrate growth /partioned Pink whitish ++ Hard/embedded 4 Round

CRF 1 Moderate growth/partioned Yellowish white + Hard/embedded 7 Round

CRF 2 Well grown/partioned Brownish Gray +++ Hard/embedded 5 Round

CRF 11 Moderate growth/partioned Yellowish white + Hard/embedded 9 Round

CRF 14 Moderate growth/partioned yellowish white + Hard/embedded 9 Round

CRF 17 Moderate growth/partioned yellowish Off white- + Hard/embedded 5 Round gray

SCF 18 Moderate growth/partioned Brownish Gray ++ Hard/embedded 5 Round

SCF 25 Moderate growth/partioned Brownish Off white ++ Hard/embedded 4 Round

SCF 31 Moderate growth/partioned Brownish Off white- ++ Hard/embedded 4 Round Gray

CTF 9 Well grown/smooth Yellowish whitish - Hard/embedded 5 Round

CTF 13 Well grown/smooth Dark Brown whitish - Hard/embedded 5 Round

CTF 14 Well grown/smooth Dark Brown Off white - Hard/embedded 7 Round

CTF 15 Well grown/smooth Dark Brown Dark gray - Hard/embedded 5 Round

CTF 20 Moderate growth/smooth Dark Brown Off white - Hard/embedded 4 Round

CTF 23 Moderate growth/smooth Dark Brown whitish + Hard/embedded 4 Round

CTF 24 Moderate growth/smooth Dark Brown whitish + Hard/embedded 4 Round

CTF 25 Moderate growth/smooth Dark Brown whitish + Hard/embedded 4 Round

Isolation and characterization of indigenous bioactive streptomycetes

Figure 3.4 Some selected Streptomyces strains exhibiting variations in morphological characteristics. A- Strain RSF18, B- Strain CRF14, C- Strain CRF17, D- Strain CTF9, E- Strain CTF15, F- Strain CTF20

Isolation and characterization of indigenous bioactive streptomycetes

A B

C D

E F

Figure 3.5 Microscopic images of some selected Streptomyces strains A- Strain RSF18 (X 500), B- Strain BG5 (X 500), C- Strain CTF9 (X 500), D- Strain CTF9 (X 1000), E- Strain SCF18 exospores (X 500), F- Strain SCF25 exospores (X 500)

Isolation and characterization of indigenous bioactive streptomycetes between 4-7 days, while in case of sugar utilization the appearance of growth was observed for two weeks.

Three out of twenty strains including CRF14, CTF23 and CTF24 did not produce melanin (Table 3.9). The formation of melanin was indicated by the appearance of a dark blue to black colour in the agar medium after incubation on melanin production medium (Table 2.11) for 7 days. The test for the utilization of eight different sugars as the sole carbon source was performed by inoculating the strains on agar slants of the basal medium (Table 2.12) containing a particular sugar as the carbon source. The appearance of growth in 7-14 days was considered as the positive result. All the strains were able to grow on glucose, fructose and mannose (Table 3.9). Only two strains BG5 and CTF20 grew on D-galactose, while three strains SCF31, CTF9, CTF13 exhibited growth on sucrose (Table 3.9). The strains CRF11, CRF14, CTF13, CTF14, CTF23 and CTF24 were found unable to utilize L-arabinose as the carbon source, however in case of raffinose six different strains showed growth on it (Table 3.9). The basal medium used for utilization of organic acids is mentioned in Tables: 2.13, 2.14, appearance of dark blue color in agar slants was observed for two weeks. It was observed that most of the strains have the ability to utilize organic acids as the dark blue spots appeared in the agar slants of all the strains except SCF18, SCF25 and SCF31. The media used for utilization of oxalates, hydrolysis of urea and hemolysis are mentioned in Tables: 2.15, 2.16, 2.17 and 2.18. In oxalates utilization clear zones around the inoculated spots of all the strains were observed on culture plates in 5-10 days except CTF23 (Table 3.10). The hydrolysis of urea was indicated as a change in colour of the basal medium from yellow to pink due to the change in pH by the production of ammonia as the byproduct of urea hydrolysis. The strains, RSF17, RSF18, RSF23, BG5, CRF1, CRF2, CRF11, CTF9, CTF13, CTF14 and CTF15 exhibited positive results in urea hydrolysis (Table 3.10). In hemolysis test only the strains SCF25, SCF31 and CTF23 showed the zones of hemolysis along the streaks on culture plates and exhibited hemolytic activity, while all other strains were found to be non hemolytic (Table 3.10).

Isolation and characterization of indigenous bioactive streptomycetes

Table: 3.8 Growth temperature range of the selected Streptomyces strains

Temperature range Strains 10˚C 20˚C 28˚C 37˚C 45˚C 55˚C 60˚C RSF 17 - + + + - - - RSF 18 - + + + - - - RSF 23 - + + + - - - BG 5 - + + + - - - CRF 1 - - + + - - - CRF 2 - - + + - - - CRF 11 - - + + - - - CRF 14 - - + + - - - CRF 17 - - + + - - - SCF 18 - - + + - - - SCF 25 - - + + - - - SCF 31 - - + + - - - CTF 9 - - + + - - - CTF 13 - - + + - - - CTF 14 - - + + - - - CTF 15 - - + + - - - CTF 20 - - + + - - - CTF 23 - - + + - - - CTF 24 - - + + - - - CTF 25 - - + + - - - (+) = growth occurred at the respective incubation temperature, (-) = no growth

Isolation and characterization of indigenous bioactive streptomycetes

Table: 3.9 Melanin production and sugar utilization profile of the selected strains.

Strains POM DG DF LA DGL RA DM SU MA

RSF 17 + + + + - + + - +

RSF 18 + + + + - + + - +

RSF 23 + + + + - + + - +

BG 5 + + + + + - + - +

CRF 1 + + + + - - + - +

CRF 2 + + + + - + + - +

CRF 11 + + + - - + + - +

CRF 14 - + + - - - - - +

CRF 17 + + + + - + + - +

SCF 18 + + + + - - + - + SCF 25 + + + + - - + - +

SCF 31 + + + + - - + + +

CTF 9 + + + + - - + + +

CTF 13 + + + - - - + + +

CTF 14 + + + - - - + - +

CTF 15 + + + + - - + - +

CTF 20 + + + + + - + - +

CTF 23 - + + - - - - - +

CTF 24 - + + - - - - - +

CTF 25 + + + + - - + - +

POM= production of melanin, DG= D-Glucose, DF= D-Fructose, LA= L-Arabinose, DGL= D-Glactose, RA= Raffinose, DM= D-Manitol, SU= Sucrose, MA= Mannose, (+) = produces malanin and utilizes the respective sugar as carbon source, (-) = no malanin production and no growth or limited growth

Isolation and characterization of indigenous bioactive streptomycetes

Table: 3.10 Results of utilization of organic acids, oxalates, hydrolysis of urea and hemolysis test of the selected Streptomyces strains

Strains Utilization of Utilization of Hydrolysis of Hemolysis

organic acids oxalate urea

RSF 17 + + + - RSF 18 + + + - RSF 23 + + + - BG 5 + + + - CRF 1 + + + - CRF 2 + + + - CRF 11 + + + - CRF 14 + + - - CRF 17 + + - - SCF 18 - + - - SCF 25 - + - + SCF 31 - + - + CTF 9 + + + - CTF 13 + + + - CTF 14 + + + - CTF 15 + + + - CTF 20 + + - - CTF 23 + - - - CTF 24 + + - + CTF 25 + + - - (+) = positive result in the respective test (-) = negative result in the respective test

Isolation and characterization of indigenous bioactive streptomycetes

DISSCUSSION

Soil is the major repository of microorganisms that produce antibiotics, for the isolation of indigenous streptomycetes we decided to collect soil samples from three different locations in our area (Table 3.1). The samples collected from botanical garden and rose fields of the university campus were organic matter rich and it was a fertile soil. These soil samples yielded the large number of Streptomyces strains and maximum isolates from this collection were found to be active in preliminary antimicrobial activity test. This may be due to the reason that this soil is arable and may also be inhabited by a large diversity of microbial flora, so the actinomycetes flora of this soil have to be more competent with respect to their antagonistic activity. The soil samples of saline farmlands also yielded a good number of bioactive Streptomyces strains; however the samples from northern areas (forest soil) yielded very small number of bioactive strains. In this study our major emphasis was on simply the collection of bioactive Streptomyces strains from our area rather than evaluation of the soil capacity to rare these bacteria and other ecological characteristics. Keeping in mind the fact that the bacteria present in different ecological niche, can exhibit varied metabolic activity and may be the potential source of new bioactive metabolites, we isolated a number of bioactive Streptomyces strains of our area. The soil samples were treated physically as well as chemically, in physical treatments the best results were obtained by prolonged heat treatment at 50˚C for three to five weeks, this strategy worked well with the samples of saline agricultural farmlands. In chemical treatment CaCO3 enrichment methods gave the best results, especially for the isolation from the soil of botanical garden and rose fields. Treatment with sodium hypochlorite found to be the useful method for isolation from the soil of northern areas (forest soil). In case of serial dilutions of the soil samples, the dilution 10-3 gave the best results for the formation of crowding on the surface of agar media. The Streptomyces colonies were selected from the crowding depending on the distinguished morphological characteristics of these filamentous bacteria as compared to the general bacteria, the colonies that were embedded into agar, had hard consistency and exhibiting variable colors, were selected preferably. At this stage only the Streptomyces colonies which were considerably different from each other in a crowding with respect to their colors or other

Isolation and characterization of indigenous bioactive streptomycetes morphological characters were selected to avoid the repetition of the strains. The selected strains were transferred to commonly used growth medium for Streptomyces (GYM agar) and were purified by repeated sub culturing to get the pure cultures. It is worth mentioning here that the original number of the strains obtained from the 30 soil samples was high and only those strains were selected finally which exhibited activity against the standard Gram positive and Gram negative test organisms in preliminary antimicrobial activity test. Solid media bioassay is an inexpensive, fast and reliable procedure for determining the antimicrobial activity of a large number of strains in short time; it provided the means to rapidly test the selected Streptomyces strains for their bioactivity and to finally select them. The finally selected Streptomyces strains (110 strains) were divided into five groups on the basis of their origin i.e. from which type of soil they have been isolated and the representative strains from each group were selected for further investigations. The selection of the representative strains from each group based on the antimicrobial activity pattern exhibited by these strains in preliminary activity analysis by solid media bioassay. In most of the cases the strains exhibiting significant activity against both the test organisms were selected and were considered for further work. The selected Streptomyces strains (20 strains) were characterized morphologically, microscopically and physiologically, the same strains were further studied in prescreening for determination of the metabolite diversity along with subsequent fermentation, purification and structural elucidation of the bioactive metabolites. The morphological characterization (Table 3.7) strongly suggests that these strains belong to the genus Streptomyces. They have hard consistency and exhibit embedded growth on the surface of agar. Both the substrate and arial mycelia show different colors including yellow, dark brown, pink, white and black, which are commonly observed in the species of the genus Streptomyces. Production of soluble pigments that diffuse into the culture medium is another typical character of the Streptomyces and perhaps of many other actinomycetes by which they can be easily recognized. The most prominent diffused pigments were observed in the cultures of the strain BG5, CRF2, SCF18, SCF25 and SCF31. The results of determination of the growth temperature range (Table 3.8) show that the strain exhibit most flourishing growth with compact hard colonies and bright

Isolation and characterization of indigenous bioactive streptomycetes colored spores mass at 28˚C (Table 3.8), which confirm that the strains in this Streptomyces collection have the optimum growth temperature in mesophylic range. The formation of melanin is a typical test which differentiates the members of the genus Streptomyces from other genera of the actinomycetales, most of the strains exhibited positive results in melanin formation which further strengthens the idea that they belong to the genus Streptomyces. In test for utilization of different sugars as carbon source (Table3.9), glucose was found to be the best compound on which all the selected strains exhibited maximum growth, along with glucose other sugars such as fructose and mannose also showed satisfactory results and most of the strains were found to be able to utilize these sugars as carbon source in the media. Other sugars such as D-glactose, sucrose, L-arabinose, raffinose etc were found to be least useful for the cultivation of these strains, as very limited growth was observed when these compounds were used as carbon source in the media (Table 3.9). Members of the genus Streptomyces are also recognized by their ability to utilize organic acids, the test was performed as agar slants and most of the strains exhibited positive results by producing blue color in the agar, only few strains were found to produce dark blue color in the media being the strongly positive. Similarly oxalate utilization was observed in almost all the strains by the appearance of clear zones around the inoculated spots of the strains in about 10 days (Table 3.10). The urease activity was exhibited by most of the strain in hydrolysis of urea test, by the appearance of pink color in the medium due to the production of ammonia in the media as a result of urea hydrolysis (Table 3.10). The comparison of the gross morphological characteristics and results of the physiological tests of these strains with those of the known actinomycetes described in Bergey’s Manual of Systematic Bacteriology (Lechevalier et al., 1989), strongly suggested that these strains belongs to the genus Streptomyces.

Biological and Chemical Screening

CHAPTER 4 PRESCREENING

In the screening for new bioactive microbial compounds, three major factors must be considered, i.e., selection of producing organisms, culture methods and detection of metabolites. The screening process from isolation of the producing microbe to recognition of a new active metabolite, earlier took up to one year (Berdy, 1985), but has shrunken to few days, using elevated methods (Laatsch, 2000). The probability of success expected for the first stage is based on the singularity of the microorganism and the novelty of both specific activity and chemical structures. In order to elucidate outstanding molecular natural diversity for drug development, various methods including target- directed biological, physico-chemical and chemical screening strategies have been developed (Fiedler, 1993; Broach and Thorner, 1996; Grabley et al., 1999). Chemical screening using TLC with various staining reagents, offers the opportunity to visualize a nearly complete picture of a microbial secondary metabolite pattern (metabolic fingerprint) (Burkhardt et al., 1996; Taddei et al., 2006). This approach can be used advantageously for both, the detection of so-called "talented" strains (Burkhardt et al., 1996), and for qualifying microbial strain collections, especially as a fundamental step of efficiently applied biological high-throughput assays. Based on their metabolic fingerprint, microbial isolates can be classified in: (i) non-producing organisms, which give no indication of the formation of secondary metabolites up to a defined detection limit under the given conditions, (ii) organisms of narrow productivity, which produce one or two secondary metabolites as main products with a restricted dependence to alteration of the culture conditions, and (iii) talented organisms, which are able to synthesize an array of structurally different secondary metabolites (Burkhardt et al., 1996). High-performance liquid chromatography (HPLC) coupled to electrospray ionization tandem mass spectrometry (ESI-MS/MS) plays an increasing role in natural product analysis, since it permits the fast screening of crude biological extracts for detailed information about metabolic profiles, with a minimum amount of material (Bringmann et al., 1999; Pelillo et al., 2004). Electrospray ionization (ESI) is the most popular technique for this purpose, as it is considered as a soft ionization technique which usually leads to

Biological and Chemical Screening only protonated or deprotonated molecules. HPLC-ESI-MS represents the combination of three powerful instruments: the HPLC and two mass spectrometers. Retention time information is obtained from the HPLC, and the mass spectrometer provides molecular mass information. If a UV detector is integrated between both instruments it generates LC/UV chromatogram, and based on UV responses, compound purity and information can be assessed as well. It is obvious that application of LC-UV/VIS-MS/MS in the chemical screening would deliver valuable supplementary information. The use of LC/MS and LC/MS/MS techniques can rapidly provide structural information of unknown trace components found in production batches. Another application of the LC/MS and LC/MS/MS technique is the identification of metabolites including reactive species in drug metabolism studies due to their sensitivity and flexibility (Kostiainen et al., 2003; Ma and Subramanian 2006). Tiller et al., 1997 have demonstrated an analytical strategy with on-line LC/UV/MS and LC/MS/MS to rapidly obtain structural information for leaches from a drug-delivery device.

The selected strains were cultivated on small scale as one liter shaking cultures and the crude extracts of the bioactive compounds obtained with ethyl acetate were screened biologically and chemically (Fig 4.1). In a biological screening the extracts were analyzed for their activity against a set of test organisms, including Gram positive bacteria (Staphylococcus aureus, Bacillus subtilis and Streptomyces viridochromogenes (Tü57), Gram negative bacteria (E. coli), fungi (Candida albicans and Mucor miehei) and microalgae (Chlorella sorokiniana, Chlorella vulgaris and Scenedesmus subspicatus) using the disc diffusion bioassay method. Additionally a cytotoxicity test was performed using a brine shrimp (Artemia salina) microwell cytotoxicity assay. In a chemical screening each of the crude extracts was analyzed by TLC (thin layer chromatography) and HPLC-MS/MS measurements. A data base search for the measured masses provided an impression of the diversity of the chemical constituents of the crude extracts produced by these strains.

In biological screening the strains were found to be active against a number of indicator micro organisms (Fig 4.2). As seen from Table 4.1, the maximum antifungal activity was exhibited by the isolates RSF23, BG5, CRF17, SCF18, SCF25, CTF9, against Candida

Biological and Chemical Screening

Prescreening 1L shaking culture

7 days shaking on an orbital shaker

Freez drying the culture broth

3X extaction with ethyle acetate

Crude extracts

Biological Chemical Screening Screening

Antimicrobial Cytotoxicity TLC HPLC-MS activity test test

Figure 4.1 Working up scheme for the pre-screening of selected Streptomyces strains.

Biological and Chemical Screening

Table 4.1 Results of screening of Streptomyces strains for antimicrobial activity and cytotoxicity

Cytotoxicity Streptomyces Antimicrobial activity test organisms (% age Strains Zone of inhibition (mm) mortality) S. virido S. B. E. C. M. C. C. S. Artemia chromogen aureus subtilus coli albicans miehei vulgaris sorokiana subspicatus salina es (Tü 57) RSF 17 14 16 13 14 + 12 - + - 15.2

RSF 18 16 17 12 15 + + - - - 13.5

RSF 23 11 - 16 - 16 + 12 11 - 92.7

BG 5 16 20 27 19 27 11 12 12 - 28.9

CRF 1 12 11 13 ------23.5

CRF 2 13 11 + ------93.7

CRF 11 22 18 17 17 - + - - - 97.2

CRF 14 18 16 18 14 - + - - - -

CRF 17 23 15 11 15 + 18 - - 25 69.3

SCF 18 - - + - + 17 - - - 78.4

SCF 25 25 22 20 20 20 + 11 12 28 91.4

SCF 31 12 - 19 + - - - - - 16.5

CTF 9 13 12 20 12 20 + 16 18 12 11.5

CTF 13 11 + 12 11 - - - - - 75.4

CTF 14 15 11 11 ------100

CTF 15 19 13 11 ------91.2

CTF 20 12 13 17 12 - + - - - 65.0

CTF 23 + + 11 ------

CTF 24 + 11 12 ------

CTF 25 11 - 15 - + - - - - 0.66 (+) = minor zone of inhibition around the disc, (-) = no activity against the test organism was observed, cytotoxicity not determined.

Biological and Chemical Screening albicans and Mucor miehei. The strains CRF17, SCF25, CTF9 were found to be highly active against microalgae (Chlorella sorokiana, Chlorella vulgaris and Scenedesmus subspicatus) (Fig 4.2). Almost all the isolates except CRF1, CRF2, and SCF18 were found to be active against Gram positive test bacteria including Staphylococcus aureus, Bacillus subtilus and Streptomyces viridochromogenes (Tü57), however the isolates RSF18, BG5, CRF11, CRF17 and SCF25 showed maximum activity against Gram negative bacteria E. coli (Table 4.1). The minimum cytotoxicity against Artimia salina was observed in case of Strains CTF25 and RSF18 while the strains SCF25, CRF2 and RSF23 were found to be highly cytotoxic (Table 4.1; Fig 4.2).

In chemical screening UV visible spots were observed on TLC plates by visualizing under UV at short and long wavelengths (254 nm, 366 nm), most of the constituents of the crude extracts exhibited UV absorbance however the most prominent UV absorbing bands were observed in the crude extracts of strains CRF2, CTF13, CTF14 and CTF15 (Fig 4.3). The pattern of colour bands after treatment of TLC plates with staining reagents

(Anisaldehyde/H2SO4 sol and Ehrlich’s reagent) is visible in Figure 4.3. The constituents of each of the crude extracts produced different colours including dark brown, blue, pink and yellow depending on the staining reagent. The most varied colour bands were observed in crude extracts of strains SCF25, CRF11, CRF14, CTF9, CTF13, CTF14, and CTF15.

In HPLC/MS analysis, each of the crude extracts exhibited a varying number of peaks at different retention times. For MS/MS measurements, a mass range of m/z = ± 1 amu was used. In most cases the quasimolecular ions were found for [M+H]+, [M+Na]+, [M-H]-, in addition to ions of dimers [2M+H]+, [2M+Na]+ and [2M-H]-, [2M+Na-2H]- etc. Sometimes the signal intensity of the dimer ion was higher than that of the monomer ion. To avoid this, an optimization was done relating to the ion source collision-induced dissociation (ISCID), which allows the dimer to decay back into the monomer. At the end collision energy around 10V was favored because of the stability of different molecules. The AntiBase search (Laatsch AntiBase, 2007) for each of the measured mass in the crude extracts of all the analyzed strains resulted in different numbers of hits. Table 4.2 summarizes the HPLC-Ms data of the selected Streptomyces strains. The crude extracts

Biological and Chemical Screening

1. 2. 3. 4.

5. 6. 7. 8.

9 10 11 12

13 14 15 16

17 18 19 20 Figure 4.2 Antimicrobial activities of selected Streptomyces strains against different test organisms: 1. Activity of strain RSF23 (1), strain BG5 (2), strain SCF25 (3) and strain CTF9 (4) against Candida albicans 2. Activity of strain RSF17 (1), strain RSF18 (2), strain SCF 18 (3) and strain CRF17 (4) against Mucor miehei 3. Activity of strain SCF31 (1), strain CRF11 (2), strain CRF2 (3) and strain CTF20 (4) against E. coli 4. Activity of strain RSF23 (1), strain BG5 (2), strain SCF25 (3) and strain CTF9 (4) against E. coli 5. Activity of strain RSF17 (1), strain RSF18 (2), strain SCF18 (3) and strain CRF17 (4) against E. coli 6. Activity of strain RSF18 (5), strain SCF18 (6) and strain CRF17 (7) against Bacillus subtilis 7. Activity of strain RSF23 (1), strain BG5 (2), strain SCF25 (3) and strain CTF9 (4) against Bacillus subtilis 8. Activity of strain SCF31 (1), strain CRF11 (2), strain CRF2 (3) and strain CTF20 (4) against Bacillus subtilis 9. Activity of strain RSF17 (1), strain RSF18 (2), strain SCF18 (3) and strain CRF17 (4) against S. aureus 10. Activity of strain RSF23 (1), strain BG5 (2), strain SCF25 (3) and strain CTF9 (4) against S. aureus 11. Activity of strain SCF31 (1), strain CRF11 (2), strain CRF2 (3) and strain CTF20 (4) against S. aureus 12. Activity of strain SCF31 (1), strain CRF11 (2), strain CRF2 (3) and strain CTF20 (4) against Streptomyces viridochromogenes (Tü57) 13. Activity of strain RSF18 (1), strain SCF 18 (2), strain CRF17 (3) and strain RSF17 (4) against Chlorella sorokiniana 14. Activity of strain RSF17 (1), strain RSF18 (2), strain SCF 18 (3) and strain CRF17 (4) against Chlorella vulgaris 15. Activity of strain RSF23 (1), strain BG5 (2), strain SCF25 (3) and strain CTF9 (4) against Chlorella sorokiana 16. Activity of strain RSF23 (1), strain BG5 (2), strain SCF25 (3) and strain CTF9 (4) against Scenedesmus subspicatus 17. Activity of strain SCF31 (1), strain CRF11 (2), strain CRF2 (3) and strain CTF20 (4) against Scenedesmus subspicatus 18. Bioautography of strain CRF17 (test organism: Staphylococcus aureus)

Biological and Chemical Screening

19. Bioautography of strain RSF18 (test organism: Staphylococcus aureus) 20. Bioautography of strain CRF14 (test organism: Staphylococcus aureus)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

B B

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

C C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

D D

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 A

Figure 4.3 Chemical screening using TLC with anisaldehyde/H2SO4 and Ehrlich’s spraying reagents. A= TLC plates under UV at 254 ηm, B= TLC plates under UV at 366 ηm, C= TLC plates after treatment with anisaldehyde/H2SO4 sol. D= TLC plates after treatment with Ehrlich’s reagent

Numbers 1-20 Crude extracts of selected strains (1=RSF17, 2=RSF18, 3=RSF23, 4=SCF18, 5=SCF25, 6=SCF31, 7=CRF1, 8=CRF2, 9=CRF11, 10=CRF14, 11=CRF17, 12=BG5, 13=CTF9, 14=CTF13, 15=CTF14, 16=CTF15, 17=CTF20, 18=CTF23, 19 =CTF24, 20=CTF25)

Biological and Chemical Screening

Table 4.2 Results of HPLC-MS/MS analysis

Retention MS Detector Mass AntiBase Strain time Rt (min) Spectrum with important peaks Assigned Search Results RSF 17 5.95 197[M+H], 196 88 hits 9.69 164.1[M+H], 163 9 hits 14.17 229[M+H], 228 70 hits 15.19 211.3[M+H], 210 38 hits 15.81 1163[M+H], 1186[M+Na]. 1162 02 hits 17.11 1132[M+H], 1154.3[M+Na]. 1131 04 hits 18.27 1116[M+H], 1138[M+Na]. 1115 10 hits 21.09 399[M+H], 416[M+18]. 398 54 hits RSF 18 1.43 329[M+Na], 635[2M+Na]. 306 97 hits 7,18 197[M+H]. 196 88 hits 8.41 164[M+H]. 163 35 hits 16.00 211[M+H]. 210 93 hits 16.32 994 [M+H], 1016 [M+Na] 993 8 hits 17.12 1132 [M+H], 1154 [M+Na] 1131 4 hits 18.32 1116 [M+H], 1138 [M+Na] 1115 10 hits 23.63 282[M+H], 563[2M+H]. 281 39 hits 26.03 908[M+H], 930[M+Na]. 907 10 hits 29.15 963[M+Na], 1903[2M+Na]. 940 2 hits RSF 23 10.10 208[M+H], 247[M+K] 207 15 hits 11.71 179[M+H], 178 42 hits 13.93 309[M+H], 308 100 hits 14.75 222[M+H], 221 13 hits 16.47 355[M+H],731[2M+H], 354 112 hits 21.42 712[M+H],734[M+Na],1445[2M+Na], 711 23 hits 22.56 740[M+H],762 [M+Na],1479[2M+H], 739 11 hits 23.71 768[M+H],790 [M+Na],1557 [2M+H], 767 8 hits 24.49 802[M+H], 1641[2M+K] 801 5 hits 25.31 1128[M+H] 1127 14 hits BG 5 8.30 224[M-H]-. 225 27 hits 10.38 583[M-H]-. 584 20 hits 10.94 599[M-H]- 600 2 hits 12.92 413[M-H]-,827[2M-H]-. 414 58 hits 13.58 650[M-H]- 651 14 hits 14.49 427[M-H], 428 33 hits 15.24 483[M-H],967[2M-H]. 484 29 hits 23.45 694[M-H]. 695 20 hits

Biological and Chemical Screening Table 4.2 continued…. 24.33 693[M-H]. 694 5 hits CRF 1 6.22 303[M+H]. 302 117 hits 7.17 261[M+H]. 260 82 hits 8.54 654[M+H],676[M+Na], 653 32 hits 1330[2M+Na]. 9.15 625[M+H],647[M+Na]. 624 18 hits 9.90 276[M-H],598[2M+HCOO-] 277 30 hits 12.91 420[M+H] 419 17 Hits 12.96 178[M-H],379[2M+Na-2H] 179 30 hits CRF 2 17.23 355[M+H], 731[2M+Na] 354 91 hits 22.43 336[M+H], 693[2M+Na] 335 21 hits 24.81 1009[M+H],1031[M+Na] 1008 15 hits 25.40 995[M+H],1017[M+Na] 994 3 hits 25.81 1023[M+H],1045[M+Na] 1022 99 hits 26.38 1009[M+H],1031[M+Na] 1008 15 hits 26.63 1037[M+H],1059[M+Na] 1036 24 hits 27.35 1023[M+H],1045[M+Na] 1022 99 hits 28.19 1037[M+H],1059[M+Na] 1036 23 hits CRF 11 17.31 353[M-H]- 354 91 hits 18.53 1329[M-H] 1330 No hits found 23.67 898[M-H], 1819[2M+Na-2H] 899 9 hits 24.81 913[M-H] 914 9 hits 26.11 880[M-H], 881 6 hits 27.77 926[M-H], 1853[2M-H] 927 8 hits CRF 14 14.83 222[M+H] 221 26hits 21.52 712[M+H], 734 [M+Na]. 711 15 hits 22.60 740[M+H], 762 [M+Na]. 739 20 hits 23.72 897[M-H] 898 19 hits 25.02 1128[M+H] 1127 17hits CRF 17 6.12 197[M+H] 196 88 hits 8.07 211[M+H] 210 93 hits 15.56 355[M+H], 731[2M+Na]. 354 91 hits 22.36 921[M+H], 1841[2M+H]. 920 3 hits 25.43 907[M+H], 1813[2M+H]. 906 10 hits SCF 18 1.80 268[M+H]. 267 35 hits 7.35 164[M+H]. 163 35 hits 7.92 211[M+H]. 210 20 hits 9.74 162[M+H]. 161 22 hits 13.32 280[M+Na], 515[2M+H]. 257 33 hits 15.32 211[M+H]. 210 93 hits

Biological and Chemical Screening Table 4.2 continued…. 15.82 355[M+H], 731[2M+Na]. 354 142 hits 17.33 381[M+H], 403[M+Na]. 380 51 hits 22.84 369[M+H]. 368 71 hits 25.43 377[M+H]. 376 69 hits SCF 25 20.71 349[M-H].721[2M+Na-2H]. 350 71 hits 23.58 898[M-H], 1795[2M-H]. 899 14 hits 23.85 782[M+H]. 781 12 hits 24.44 796[M+H],1613[2M+Na]. 795 24 hits 24.05 922[M+Na] 899 6 hits 24.06 912[M-H], 1825[2M-H]. 913 10 hits 26.74 884[M-H], 1769[2M-H]. 885 13 hits SCF 31 13.92 166[M+H]. 165 17 hits 16.93 960[M+H]. 959 7 hits 18.61 1074[M+H]. 1073 5 hits 19.41 1187[M+H]. 1186 3 hits 24.20 579[M+H], 601[M+Na]. 578 22 hits 28.47 908[M+H], 930[M+Na]. 907 10 hits CTF 9 5.54 143[M+H] 142 23 hits 6.98 197[M+H] 196 23 hits 8.23 226[M+H] 225 87 hits 8.62 240[M+H] 239 21 hits 9.13 585[M+H] 584 32 hits 14.08 309[M+H] 308 24 hits 15.86 959[M+H] 958 5 hits 16.77 323[M+18] 305 16hits 23.91 787[M+H] 786 21 hits 24.52 802[M+H] 801 15 hits CTF 13 16.95 355[M-H], 733[2M+Na-2H]. 356 91 hits 17.30 355[M+H], 731[2M+Na]. 354 91 hits 20.59 317[M-H], 339[M+Na-2H]. 318 143 hits 22.57 335[M-H], 693[2M+Na-2H]. 336 139 hits 25.41 863[M+H] 862 3 hits 28.30 377[M+H],75[2M+Na]. 376 51 hits CTF 15 7.15 227[M+H], 249[M+Na] 226 70 hits 8.10 243[M+H], 265[M+Na] 242 78 hits 14.43 280[M+18] 262 131 hits 16.33 304[M+H] 303 16 hits 16.78 211[M+H] 210 91 hits 17.35 355[M+H],731[2M-Na+2H] 354 77 hits 18.43 311[M-H] 312 78 hits

Biological and Chemical Screening Table 4.2 continued…. 20.53 339[M-H],701[2M-Na+2H] 340 74 hits 22.25 391[M-H], 805[2M-Na+2H] 392 62 hits 22.55 335[M-H], 693[2M-Na+2H] 336 97 hits 23.80 377[M-H] 378 51 hits 24.28 516[M+H] 515 31 hits 24.94 319[M-H] 320 131 hits 25.68 592[M+H],1160[2M+Na] 591 28 hits 28.82 377[M+H], 775[2M+Na] 376 69 hits CTF 23 25.54 994[M+H], 1017[M+Na]. 993 8 hits 26.21 1008[M+H], 1030[M+H]. 1007 8 hits 26.82 1058[M+Na]. 1035 12 hits 27.46 1044[M+Na]. 1021 9 hits 27.78 1020[M-H] 1022 9 hits 28.44 1036[M+H], 1058[M+Na]. 1035 12 hits 28.66 1034[M-H] 1035 12 hits CTF 24 5.77 298[M+H] 297 79 hits 6.81 261[M+H]. 260 82 hits 11.89 164[M+H] 163 35 hits 16.60 211[M+H] 210 93 hits 17.50 332[M+H] 331 30 hits 18.71 360[M+H]. 359 24 hits 20.65 1651[M-H] 1652 2 hits 21.03 936[M+H] 935 3 hits 29.49 964[M+H] 963 4 hits CTF 25 6.78 157[M+H] 156 49 hits 14.39 280[M+H] 279 21 hits 16.84 869[M+H] 868 7 hits 17.11 884[M+H] 883 3 hits 19.20 1188[M+H] 1187 7 hits 22.79 920[M+H] 919 3 hits 23.96 922[M+H] 921 8 hits 25.34 936[M+H] 935 3 hits 27.80 858[M+H] 857 7 hits Selected ion in each of the extract for which HPLC-Ms chromatogram is given is highlighted

Biological and Chemical Screening

RT: 0,00 - 30,00 18,81 23,83 NL: 100 4,10E7 90 Base Peak MS 80 RSF17dp1

70 8,17 8,29 60

50 14,24 8,53 40 17,11 23,91 15,19 RelativeAbundance 30 5,95 7,34 9,69 9,78 20 5,63 9,91 21,09 1,11 13,78 10 1,04 2,98 25,21 27,23 0 1,03 NL: 200000 2,04E5 9,04 Total Scan 180000 17,07 PDA 160000 1,44 RSF17dp1

140000

120000 6,80 6,58 100000 3,55 12,32 18,19 uAU 5,51 7,09 9,85 80000 14,16 11,30 60000 18,72 40000 19,32 20,52 20000 23,72 24,71 27,17 0 0 5 10 15 20 25 30 Time (min)

Figure 4.4a Total ion chromatogram of crude extract RSF17

I:\DATABASE\...\Imra\Imransajid\RSF17dp1 23.02.2007 04:50:14

RT: 15,53 - 19,17 17,11 NL: 100 1,45E7 17,20 Base Peak m/ z= 1131,5-1132,5 50 17,29 MS RSF17dp1 17,02 17,66 17,75 15,70 15,87 15,95 16,04 16,30 16,48 16,66 16,84 17,37 17,85 18,02 18,11 18,36 18,61 18,75 18,81 19,06 0 17,07 NL: 1,77E5 150000 Tot al Scan PDA 18,19 100000 RSF17dp1 15,94 uAU 15,82 15,56 16,14 50000 16,31 16,46 17,44 17,61 17,76 18,48 18,72

0 15,6 15,8 16,0 16,2 16,4 16,6 16,8 17,0 17,2 17,4 17,6 17,8 18,0 18,2 18,4 18,6 18,8 19,0 Time (min)

RSF17dp1 #697 RT: 17,11 AV: 1 NL: 1,45E7 T: + c ESI sid=10,00 Full ms [ 100,00-2000,00] 1132,1 100 1154,3

20 1156,2 Relative Abundance 261,3 345,2 495,4 246,2 401,1 511,8 679,4 751,8 879,8 928,2 1086,3 1171,7 1284,3 1361,7 1505,5 1556,3 1645,7 1730,1 1781,5 1906,2 1960,3 0 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z

RSF17dp1 #698 RT: 17,14 AV: 1 NL: 3,10E5 T: + c sid=10,00 d Full ms2 1132,06@35,00 [ 300,00-2000,00] 746,1 100 1114,0 798,1 907,0 80

964,9 1074,1 60 895,0

40 631,0 1017,2 579,0 851,1 20 694,2 Relative Abundance 370,2 463,9 481,6 1115,7 1698,2 0 400 600 800 1000 1200 1400 1600 1800 2000 m/z

Figure 4.4b Selected ion chromatogram of extract RSF17 (first line), UV chromatogram, mass spectrum of the signal at Rt = 17.11 min, and MS/MS fragmentation (bottom) of the ion signal [M+Na] at m/z 1154.

Biological and Chemical Screening

RT: 0,00 - 30,06 24,89 NL: 100 5,12E7 90 Base Peak MS 80 RSF18dp1 70 9,28 60 9,58 50 16,00 20,06 17,12 40 24,81 8,41 9,70 18,32 25,05 7,18 11,24 15,78 RelativeAbundance 30 26,03 20 1,05 6,99 21,59 3,77 10 11,75 15,50 29,15 0 1,35 NL: 3,45E5 Total Scan 300000 17,05 PDA RSF18dp1 250000 18,24 200000

10,03 uAU 150000 5,65 1,79 6,51 16,23 100000 7,98 13,75 3,55 10,96

50000 19,98 20,24 22,43 24,83 27,84 0 0 5 10 15 20 25 30 Time (min)

Figure 4.5a Total ion chromatogram of crude extract RSF18

Figure 4.5b Selected ion chromatogram of extract RSF18 (first line), UV chromatogram, mass spectrum of the signal of 1 at Rt = 17.12 min, and MS/MS fragmentation (bottom) of the ion signal [M+Na] at m/z 1154.

Biological and Chemical Screening

RT: 0,00 - 30,00 22,56 NL: 100 6,92E7 90 Base Peak 22,63 MS 80 RSF23dp1 21,41 70

60 22,86 14,75 50 9,40 25,28 9,10 9,46 24,33 40 16,47 10,10 19,38 RelativeAbundance 30 13,96 11,75 6,93 6,87 17,05 20 1,46 13,17 28,33 28,98 6,11 7,12 5,52 10 3,47

0 9,86 NL: 8,59 11,72 14,72 5,29E5 500000 Total Scan 16,35 PDA 450000 1,35 13,59 18,45 RSF23dp1 400000 18,76 350000 5,39 7,75 300000 6,21 22,13 22,49

uAU 250000 1,75 200000 22,73 4,21 150000 23,64 100000 25,01 50000 0 0 5 10 15 20 25 30 Time (min)

Figure 4.6a Total ion chromatogram of crude extract RSF23

I:\DATABASE\...\Imra\Imransajid\RSF23dp1 14.03.2007 18:57:29

RT: 0,00 - 30,00 22,56 NL: 100 6,92E7 22,63 21,41 Base Peak 22,86 MS 9,40 14,75 25,28 RSF23dp1 50 9,10 9,46 23,71 15,19 16,47 19,38 8,97 11,75 13,96 21,17 1,46 6,63 6,87 10,91 13,17 17,05 28,33 28,98 0,77 3,41 3,65 5,52 27,48 0 9,86 NL: 8,59 11,72 14,72 5,29E5 13,59 16,35 1,35 10,19 13,13 18,45 Tot al Scan 400000 18,76 5,39 7,75 PDA 6,21 19,29 22,13 22,49 RSF23dp1

uAU 1,75 22,73 200000 1,22 4,21 23,64 3,49 25,01 29,34 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Time (min)

RSF23dp1 #922 RT: 21,41 AV: 1 NL: 4,93E7 T: + c ESI sid=10,00 Full ms [ 100,00-2000,00] 734,4 100 712,3

60 1446,2 40 735,5 1444,8 20 Relative Abundance 266,0 771,3 1087,6 1476,1 207,9 282,0 400,8 498,4 615,8 695,0 805,4 1019,8 1197,9 1306,7 1521,9 1699,7 1734,7 1935,2 0 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z

RSF23dp1 #924 RT: 21,46 AV: 1 NL: 5,45E6 T: + c sid=10,00 d Full ms2 712,32@35,00 [ 185,00-1435,00] 371,9 100

80 341,2 60 323,1 40 390,0

20 Relative Abundance 305,0 584,1 205,0 390,9 571,6 694,3 710,2 0 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z

Figure 4.6b Selected ion chromatogram of extract RSF23 (first line), UV chromatogram, mass spectrum of the signal at Rt = 21.41 min, and MS/MS fragmentation (bottom) of the ion signal [M+Na] at m/z 734. 98

Biological and Chemical Screening

RT: 0,00 - 30,03 24,33 NL: 100 2,84E7 90 Base Peak MS 80 BG5dn1 24,12 70 24,40 23,45 60

50 24,85 26,42 40 10,94 22,75

RelativeAbundance 30 10,38 22,47 12,92 15,24 20 21,88 10,14 8,30 14,49 15,33 20,68 10 6,78 3,50 4,55 6,37 0 12,28 NL: 500000 5,06E5 11,56 Total Scan 450000 13,61 18,11 PDA 400000 1,35 11,31 BG5dn1 15,39 350000 5,34 18,38 20,42 300000 8,78

250000 8,25 uAU 200000 1,73 21,08 4,15 150000 21,42 29,11 100000 23,84 25,05 50000

0 0 5 10 15 20 25 30 Time (min)

Figure 4.7a Total ion chromatogram of crude extract BG5

I:\DATABASE\...\Imra\Imransajid\BG5dn1 14.03.2007 23:02:26

RT: 0,00 - 30,03 24,33 NL: 100 2,84E7 24,12 Base Peak 23,45 MS BG5dn1 50 10,94 22,75 24,85 26,42 27,53 29,61 10,38 12,92 15,24 22,47 8,30 11,63 14,49 15,33 21,36 0,10 1,42 3,23 4,15 4,55 6,78 8,23 9,46 18,32 19,32 0 12,28 NL: 11,56 13,61 18,11 5,06E5 400000 1,35 15,39 Tot al Scan 5,34 14,72 17,09 18,38 PDA BG5dn1 8,78 10,45 20,42

1,73 6,15 8,25 21,08 uAU 200000 1,21 3,83 4,15 23,13 29,11 23,84 25,05 28,37 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Time (min)

BG5dn1 #905 RT: 24,33 AV: 1 NL: 2,84E7 T: - c ESI sid=10,00 Full ms [ 100,00-2000,00] 693,5 100

20 Relative Abundance 333,4 209,4 245,4 423,6 516,3 676,6 705,6 862,9 967,9 1044,5 1113,6 1230,7 1302,6 1387,5 1478,7 1590,7 1698,9 1741,8 1861,4 1942,5 0 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z

BG5dn1 #906 RT: 24,36 AV: 1 NL: 3,19E6 T: - c sid=10,00 d Full ms2 693,52@35,00 [ 180,00-1400,00] 377,2 100

60 451,3 40 469,3 241,4 693,5

20 Relative Abundance 227,2 391,2 470,2 619,6 695,2 0 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z

Figure 4.7b Selected ion chromatogram of extract BG5 (first line), UV chromatogram, mass spectrum of the signal at Rt = 24.33 min, and MS/MS fragmentation (bottom) of the ion signal [M+Na] at m/z 693.

Biological and Chemical Screening

RT: 0,00 - 30,02 26,40 NL: 100 1,78E7 90 Base Peak MS 80 crf1dp1

70 26,32 60 26,56

50 10,14 10,40 40 10,47 8,54 RelativeAbundance 30 10,60 26,24 5,86 7,93 12,91 20,79 20 5,79 16,89 21,60 26,72 12,99 18,54 23,50 10 25,13 27,07 1,04 2,83 4,55 0 1,40 NL: 11,65 1,18E5 Total Scan 100000 PDA 10,74 crf1dp1 12,57 80000 6,19 10,44 7,86 60000 9,19

uAU 13,36 20,68 14,75 16,86 40000 4,33 5,82 17,97 26,35 3,72 21,42 2,26 22,37 20000 27,07

0 0 5 10 15 20 25 30 Time (min)

Figure 4.8a Total ion chromatogram of crude extract CRF1

I:\DATABASE\Neue Daten\Imra\crf1dp1 06.06.2007 17:43:14

RT: 0,00 - 30,02 26,40 NL: 100 1,78E7 Base Peak 26,56 MS crf 1dp1 50 10,14 10,40 8,54 26,24 5,79 5,86 7,93 10,73 12,91 16,89 18,54 20,79 21,60 23,50 26,72 1,04 1,52 2,83 4,55 9,72 12,99 15,01 19,90 29,40 0 1,40 NL: 11,65 1,18E5 100000 10,74 Tot al Scan 1,25 12,57 12,78 6,19 6,66 7,43 10,44 PDA crf 1dp1 13,36 14,75 16,86 20,68 uAU 50000 5,82 14,91 3,72 4,33 17,97 18,42 26,35 2,26 21,42 22,37 23,15 27,07 28,24 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Time (min)

crf1dp1 #493 RT: 12,91 AV: 1 NL: 3,46E6 T: + c ESI sid=10,00 Full ms [ 100,00-2000,00] 420,3 100

40 421,3 20 227,3

Relative Abundance 283,2 213,1 564,2 613,3 661,2 771,4 816,7 866,4 1059,1 1210,9 931,7 1253,5 1361,5 1525,0 1641,1 1823,2 1870,2 1899,1 0 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z

crf1dp1 #494 RT: 12,95 AV: 1 NL: 9,26E5 T: + c sid=10,00 d Full ms2 420,30@35,00 [ 105,00-855,00] 305,1 100

40 388,3 306,1 20 Relative Abundance 419,2 122,8 167,1 189,1 226,9 274,2 288,1 312,4 376,2 447,1 451,7 582,3 0 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 m/z

Figure 4.8b Selected ion chromatogram of extract CRF1 (first line), UV chromatogram, mass spectrum of the signal at Rt = 12.91 min, and MS/MS fragmentation (bottom) of the ion signal [M+H] at m/z 420.

100

Biological and Chemical Screening

RT: 0,00 - 30,07 25,73 NL: 100 5,07E7 26,71 90 Base Peak 27,35 MS 80 crf2dp1

60 28,26

50 24,89

RelativeAbundance 30

20 16,73 14,33 17,30 21,45 10 9,77 12,68 9,85 14,79 17,38 23,98 1,45 4,23 6,40 7,68 0 10,73 NL: 2,91E5 22,23 Total Scan 250000 PDA crf2dp1 28,21 200000

150000

uAU 11,08 8,57 100000 6,97 12,56 15,71 1,39 20,58 14,27 16,67 50000 5,98 6,28 17,68 22,66 23,98 3,53 5,27 0 0 5 10 15 20 25 30 Time (min)

Figure 4.9a Total ion chromatogram of crude extract CRF2

I:\DATABASE\Neue Daten\Imra\crf2dp1 06.06.2007 20:26:29

RT: 0,00 - 30,07 25,73 NL: 100 26,71 5,07E7 27,35 25,65 Base Peak 28,26 MS crf 2dp1 50 24,89 28,34 28,59 14,33 16,73 17,30 20,63 21,45 0,56 1,45 3,52 4,23 5,57 6,40 7,68 8,41 9,05 9,77 10,14 12,68 14,79 18,81 21,71 23,98 29,51 0 10,73 NL: 22,23 2,91E5 28,21 Tot al Scan 200000 PDA crf2dp1 11,08

uAU 8,57 6,97 15,71 100000 1,39 10,21 12,56 14,27 16,67 17,21 20,58 5,98 6,28 7,63 18,18 22,66 23,49 3,53 4,22 26,32 29,31 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Time (min)

crf2dp1 #1015 RT: 28,18 AV: 1 NL: 2,26E7 T: + c ESI sid=10,00 Full ms [ 100,00-2000,00] 1058,9 100

40 1036,6 20 Relative Abundance 538,3 1080,7 204,7 347,1 377,3 478,9 703,5 756,3 881,5 945,3 1022,7 1188,0 1375,8 1399,5 1449,5 1598,5 1755,0 1780,8 1936,4 0 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z

crf2dp1 #1016 RT: 28,21 AV: 1 NL: 7,98E6 T: + c sid=10,00 d Full ms2 1058,86@35,00 [ 280,00-2000,00] 814,5 100 707,5 80 1058,7 60 1059,7 815,6 40 945,6 20 463,3 1040,6 Relative Abundance 594,4 717,4 828,5 367,1 391,4 464,2 699,4 1076,7 0 400 600 800 1000 1200 1400 1600 1800 2000 m/z

Figure 4.9b Selected ion chromatogram of extract CRF2 (first line), UV chromatogram, mass spectrum of the signal at Rt = 28.18 min, and MS/MS fragmentation (bottom) of the ion signal [M+Na] at m/z 1059.

101

Biological and Chemical Screening

RT: 0,00 - 30,00 23,83 NL: 100 1,12E7 23,75 90 24,06 Base Peak MS 80 crf11dn1

70 24,22

60 24,81 50 25,47 27,77 40 26,19 27,85

RelativeAbundance 30 14,05 27,94 20 18,53 23,41 10 13,44 14,34 17,39 23,13 6,93 20,41 1,20 3,90 6,80 10,15 11,07 0 10,83 NL: 4,40E5 400000 Total Scan PDA 350000 crf11dn1

300000

250000 6,03 14,00

uAU 200000 14,44 6,99 11,16 150000 10,29 7,23 100000 1,39 16,70 17,26 1,86 5,54 20,68 50000 21,81 22,69 24,85 27,80 0 0 5 10 15 20 25 30 Time (min)

Figure 4.10a Total ion chromatogram of crude extract CRF11

I:\DATABASE\Neue Daten\Imra\crf11dn1 07.06.2007 03:55:22

RT: 0,00 - 30,00 23,83 NL: 100 24,06 1,12E7 Base Peak 24,22 MS crf 11dn1 24,81 50 23,59 25,47 27,77 14,05 26,27 28,02 14,34 18,53 23,41 1,20 2,81 3,90 5,41 6,80 6,93 7,42 9,07 10,15 11,07 11,92 13,44 17,31 17,39 20,41 21,83 0 10,83 NL: 4,40E5 400000 Tot al Scan PDA 6,03 6,20 14,00 crf 11dn1 10,65 11,16 14,44 uAU 200000 11,97 1,39 7,23 8,86 9,51 15,75 16,70 17,26 1,19 1,86 4,25 5,54 18,45 20,68 21,81 22,11 23,48 24,85 26,48 27,80 28,80 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Time (min)

crf11dn1 #693 RT: 18,53 AV: 1 NL: 1,59E6 T: - c ESI sid=10,00 Full ms [ 100,00-2000,00] 1329,4 100

40 1223,5 1331,6 20 Relative Abundance 1180,7 479,5 761,4 1115,6 1225,7 1376,2 1584,3 1711,4 231,5 270,1 453,7 615,8 671,8 893,9 1033,2 1550,4 1862,3 1992,0 0 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z

crf11dn1 #694 RT: 18,56 AV: 1 NL: 7,67E5 T: - c sid=10,00 d Full ms2 1329,41@35,00 [ 355,00-2000,00] 1223,7 100

20 Relative Abundance 1179,7 591,3 653,5 731,6 798,5 910,4 975,8 1099,6 1161,6 1285,6 1311,6 0 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 m/z

Figure 4.10b Selected ion chromatogram of extract CRF11 (first line), UV chromatogram, mass spectrum of the signal at Rt = 18.53 min, and MS/MS fragmentation (bottom) of the ion signal [M-H] at m/z 1329.

102

Biological and Chemical Screening

RT: 0,00 - 30,00 23,72 NL: 100 3,14E7 90 23,65 Base Peak MS 80 23,88 crf14adn1

70 24,74 60 25,29 50 27,58 40 25,37

RelativeAbundance 30 27,74

20 14,15 23,24 14,27 10 13,51 22,89 1,10 2,37 5,15 7,05 10,43 11,14 17,37 17,77 0 10,88 NL: 3,97E5 Total Scan 350000 PDA crf14adn1 300000

250000 6,10 14,08 200000 uAU 14,36 13,42 150000 7,06 12,03 1,39 10,35 100000 7,29 16,76 1,85 17,68 20,71 4,28 50000 21,53 22,73 24,82 28,59 0 0 5 10 15 20 25 30 Time (min)

Figure 4.11a Total ion chromatogram of crude extract CRF14

I:\DATABASE\Neue Daten\Imra\crf14adn1 07.06.2007 01:12:10

RT: 0,00 - 30,00 23,72 NL: 100 23,80 3,14E7 Base Peak 23,57 24,11 24,82 MS crf 14adn1 50 27,58 25,45 27,74 14,15 23,33 28,59 0,51 1,89 2,37 3,57 5,15 6,36 7,05 8,41 10,43 11,14 11,70 13,51 14,44 17,15 17,37 18,60 20,36 21,79 22,89 0 10,88 NL: 3,97E5 Tot al Scan 6,10 14,08 PDA crf 14adn1 200000 14,36 uAU 7,06 10,70 11,22 13,42 1,39 7,29 9,57 16,76 1,19 1,85 4,28 5,61 15,83 17,68 19,53 20,71 20,98 3,03 22,73 23,73 25,96 26,37 28,59 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Time (min)

crf14adn1 #880 RT: 23,72 AV: 1 NL: 3,14E7 T: - c ESI sid=10,00 Full ms [ 100,00-2000,00] 897,7 100

20 899,8 1796,3 Relative Abundance 865,8 254,4 363,3 425,5 465,6 583,0 693,7 760,4 912,6 960,5 1048,1 1173,0 1233,4 1406,1 1451,7 1591,5 1622,0 1714,1 1817,5 1913,1 1957,1 0 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z

crf14adn1 #881 RT: 23,75 AV: 1 NL: 2,09E7 T: - c sid=10,00 d Full ms2 897,70@35,00 [ 235,00-1810,00] 865,6 100

40 847,7

20 Relative Abundance 425,6 565,8 597,7 829,7 879,6 1705,0 0 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 m/z

Figure 4.11b Selected ion chromatogram of extract CRF14 (first line), UV chromatogram, mass spectrum of the signal at Rt = 23.72 min, and MS/MS fragmentation (bottom) of the ion signal [M-H] at m/z 897.

103

Biological and Chemical Screening

RT: 0,00 - 30,00 25,43 NL: 100 3,31E8 90 Base Peak MS 80 CRF17dp1 25,29 70

40 23,97 22,36

RelativeAbundance 30 22,12 20 18,73 8,07 8,01 21,09 10 1,91 2,55 15,06 15,56 27,29 6,12 9,77 9,93 17,39 0 1,05 NL: 2,65E5 250000 1,40 9,18 Total Scan PDA CRF17dp1 200000 4,05

150000 12,15

5,30 5,70 9,68 uAU

100000 8,78 9,98 7,48

50000 13,03 15,50 18,68 19,69 23,91 24,35 27,47 0 0 5 10 15 20 25 30 Time (min)

Figure 4.12a Total ion chromatogram of crude extract CRF17

I:\DATABASE\...\Imra\Imransajid\CRF17dp1 23.02.2007 04:09:28

RT: 0,00 - 30,00 25,43 NL: 100 25,35 3,31E8 25,52 Base Peak 25,60 MS CRF17dp1 50 23,97 24,97 22,36 22,52 8,07 18,73 19,61 21,09 1,72 1,91 2,55 3,10 5,80 6,12 7,95 8,44 9,77 11,88 12,83 15,06 15,56 15,63 27,29 28,61 0 1,05 NL: 1,40 9,18 2,65E5 Tot al Scan 200000 3,64 4,05 12,15 PDA 5,30 5,70 9,68 CRF17dp1

uAU 8,78 9,98 100000 6,06 7,48 11,36 13,03 14,99 15,50 18,68 15,92 19,69 20,61 21,24 23,91 24,35 25,88 27,47 28,24 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Time (min)

CRF17dp1 #928 RT: 22,36 AV: 1 NL: 9,98E7 T: + c ESI sid=10,00 Full ms [ 100,00-2000,00] 921,6 100

60 831,4 922,6 40 916,4 20 936,6

Relative Abundance 813,4 185,3 205,8 345,4 505,6 558,1 691,4 802,3 945,0 1109,6 1156,1 1360,9 1412,6 1526,4 1627,5 1774,3 1841,1 1884,6 1992,2 0 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z

CRF17dp1 #929 RT: 22,38 AV: 1 NL: 3,39E7 T: + c sid=10,00 d Full ms2 921,55@35,00 [ 240,00-1855,00] 889,5 100

40 871,6

20 903,4 Relative Abundance 489,5 521,6 659,1 695,3 853,6 0 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 m/z

Figure 4.12b Selected ion chromatogram of extract CRF17 (first line), UV chromatogram, mass spectrum of the signal at Rt = 22.36 min, and MS/MS fragmentation (bottom) of the ion signal [M+H] at m/z 921.

104

Biological and Chemical Screening

RT: 0,00 - 30,00 23,85 NL: 100 5,91E7 90 Base Peak MS 80 23,95 SCF18dp1

70 18,86 7,92 60 8,28

50 22,84 40 15,82 8,47 13,23 RelativeAbundance 30 9,74 22,65 1,80 5,68 7,35 20 2,44 9,96 17,33 24,03 20,58 25,43 3,00 11,40 10 27,29 0 9,13 NL: 450000 4,56E5 Total Scan 400000 PDA SCF18dp1 350000

300000

250000 1,04 1,76 4,00 uAU 200000 6,84 6,62 25,36 150000 8,70 9,94 100000 10,22 11,35 21,10 15,78 19,45 50000 21,86 27,19 0 0 5 10 15 20 25 30 Time (min)

Figure 4.13a Total ion chromatogram of crude extract SCF18

I:\DATABASE\...\Imra\Imransajid\SCF18dp1 23.02.2007 06:52:41

RT: 0,00 - 30,00 23,85 NL: 100 5,91E7 23,95 Base Peak 7,92 18,86 MS 7,86 8,28 SCF18dp1 50 22,84 13,23 15,32 15,82 15,89 1,80 8,53 9,74 13,05 22,65 1,20 2,44 5,56 5,68 7,35 15,19 17,33 21,31 24,03 3,00 10,02 12,00 16,61 25,43 27,29 28,26 0 9,13 NL: 4,56E5 400000 Tot al Scan PDA 1,04 1,76 3,64 4,00 SCF18dp1 6,62 6,84 25,36 uAU 200000 8,70 9,94 10,22 21,10 11,79 13,21 15,23 15,78 18,84 19,45 18,06 21,86 23,82 27,19 28,01 28,89 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Time (min)

SCF18dp1 #679 RT: 15,82 AV: 1 NL: 2,10E7 T: + c ESI sid=10,00 Full ms [ 100,00-2000,00] 730,9 100

80 355,2

Relative Abundance 757,0 211,1 299,0 377,0 1084,4 193,0 419,2 501,2 708,3 786,1 869,9 937,2 1167,3 1256,2 1399,2 1543,9 1635,3 1727,3 1809,3 1888,0 1996,6 0 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z

SCF18dp1 #680 RT: 15,85 AV: 1 NL: 6,21E5 T: + c sid=10,00 d Full ms2 730,94@35,00 [ 190,00-1475,00] 377,1 100

20 Relative Abundance 242,8 403,6 491,4 582,2 611,5 699,1 712,2 0 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z

Figure 4.13b Selected ion chromatogram of extract SCF18 (first line), UV chromatogram, mass spectrum of the signal at Rt = 15.82 min, and MS/MS fragmentation (bottom) of the ion signal [M+H] at m/z 355.

105

Biological and Chemical Screening

RT: 0,00 - 30,05 19,28 NL: 100 9,78 5,61E6 90 14,35 Base Peak MS 80 9,99 20,68 ctf25dp1 9,71 70 14,28 16,75 10,13 60 6,93 50 19,00 25,55 23,96 26,49 5,62 9,04 20,89 40 10,27 18,70 11,93 RelativeAbundance 30 5,47 27,80 1,44 27,93 20 5,31 14,50 28,19 4,16 10

0 6,98 NL: 160000 1,61E5 1,38 Total Scan 140000 PDA 5,98 ctf25dp1 120000 10,76 8,64 100000 11,10 12,38

80000 13,54 uAU 14,22 60000 16,65 20,61 5,54 17,22 40000 21,45 4,31 22,68 3,28 26,42 20000 28,68

0 0 5 10 15 20 25 30 Time (min)

Figure 4.14a Total ion chromatogram of crude extract SCF25

I:\DATABASE\Neue Daten\Imra\ctf25dp1 07.06.2007 02:33:46

RT: 0,00 - 30,05 NL: 9,78 19,28 100 14,35 5,61E6 9,99 14,28 20,68 Base Peak 16,75 MS ctf25dp1 6,93 16,67 19,00 23,96 25,55 50 5,62 8,33 9,04 10,20 19,57 20,89 23,18 26,49 5,47 11,93 18,61 27,80 1,04 1,44 13,64 20,96 1,52 4,16 14,50 28,19 0 6,98 NL: 1,38 1,61E5 150000 5,98 Tot al Scan 1,20 10,76 8,64 11,10 PDA 100000 8,10 10,23 11,81 13,54 ct f 25dp1

uAU 14,22 16,65 20,61 50000 5,54 17,22 18,27 2,28 3,28 4,31 21,45 21,96 24,34 26,42 28,68 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Time (min)

ctf25dp1 #865 RT: 24,05 AV: 1 NL: 2,33E6 T: + c ESI sid=10,00 Full ms [ 100,00-2000,00] 921,7 100

60 922,7

20 301,1 907,6 Relative Abundance 204,9 401,2 547,9 630,2 653,5 717,4 857,6 937,4 1117,5 1162,6 1292,6 1390,6 1441,5 1636,3 1743,5 1828,6 1904,1 1940,9 0 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z

ctf25dp1 #866 RT: 24,08 AV: 1 NL: 2,22E6 T: + c sid=10,00 d Full ms2 921,70@35,00 [ 240,00-1855,00] 889,7 100

20 Relative Abundance 354,4 386,7 535,4 595,1 713,1 818,4 857,7 903,5 0 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 m/z

Figure 4.14b Selected ion chromatogram of extract SCF25 (first line), UV chromatogram, mass spectrum of the signal at Rt = 24.05 min, and MS/MS fragmentation (bottom) of the ion signal [M+Na] at m/z 922.

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Biological and Chemical Screening

RT: 0,00 - 30,02 20,86 NL: 100 4,13E6 90 Base Peak MS 80 scf31dp1

70 21,00 60

50 10,41 26,25 10,33 21,14 40 17,02 13,84 24,20 RelativeAbundance 21,28 30 28,43 10,64 20,73 21,51 20 9,71 14,61 3,70 7,20 7,92 23,04 2,16 11,04 15,93 10

0 10,99 NL: 6,38E4 60000 Total Scan PDA 13,80 50000 20,76 scf31dp1

40000 10,77

1,41 7,24

uAU 30000 12,06 16,93 10,48 14,48 17,44 21,51 6,30 9,68 20000 22,97 24,12 26,14 27,83 5,87 10000 4,34

0 0 5 10 15 20 25 30 Time (min)

Figure 4.15a Total ion chromatogram of crude extract SCF31

I:\DATABASE\Neue Daten\Imra\scf31dp1 06.06.2007 18:24:04

RT: 0,00 - 30,02 20,86 NL: 100 4,13E6 Base Peak MS scf 31dp1 10,41 50 10,33 21,14 26,25 13,84 16,83 17,02 24,20 26,33 28,43 7,92 9,71 10,64 17,11 18,61 20,73 21,51 21,97 1,04 2,16 3,70 4,77 4,93 7,20 11,36 15,93 24,29 0 10,99 NL: 60000 6,38E4 13,80 20,76 Tot al Scan 10,77 PDA 40000 1,41 7,24 scf 31dp1 10,48 12,06 14,48 14,74 16,93 uAU 6,30 9,68 17,44 18,43 21,51 8,28 21,97 22,97 24,12 26,14 20000 1,24 5,87 26,92 28,78 1,97 3,81 4,34 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Time (min)

scf31dp1 #703 RT: 19,41 AV: 1 NL: 7,97E5 T: + c ESI sid=10,00 Full ms [ 100,00-2000,00] 1187,7 100

40 1329,9 1189,8 1536,2 20 271,6 383,3 848,5 1398,6 1642,1 Relative Abundance 149,0 470,5 608,3 642,1 803,3 918,4 945,7 1097,2 1257,4 1466,2 1671,6 1790,1 1904,0 1986,4 0 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z

scf31dp1 #704 RT: 19,44 AV: 1 NL: 2,47E5 T: + c sid=10,00 d Full ms2 1187,71@35,00 [ 315,00-2000,00] 1169,7 100

80 959,6 60 1170,7 40 960,6 1151,6

20 941,7 1127,7 Relative Abundance 536,3 863,2 1097,8 437,7 649,5 737,7 1003,5 1188,9 1644,2 0 400 600 800 1000 1200 1400 1600 1800 2000 m/z

Figure 4.15b Selected ion chromatogram of extract SCF31 (first line), UV chromatogram, mass spectrum of the signal at Rt = 19.41 min, and MS/MS fragmentation (bottom) of the ion signal [M+H] at m/z 1187.

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Biological and Chemical Screening

RT: 0,00 - 30,00 9,13 NL: 9,44 100 3,81E7 90 Base Peak 9,56 MS 80 24,52 25,31 CTF9dp1 70 23,91 15,94 8,69 60 16,77 50 9,81 6,98 25,40 9,93 14,08 40 6,79 13,48 5,54 10,06 25,52 RelativeAbundance 30 18,08 5,24 20,54 25,60 28,95 20 3,66 11,85 1,14 23,68 10

0 9,92 NL: 500000 5,05E5 1,35 450000 Total Scan 13,70 PDA 400000 8,64 10,27 14,53 CTF9dp1

350000 5,40 6,23 14,84 16,04 300000 16,92 250000 17,45 uAU 1,63 4,19 200000 20,57

150000 21,55 100000 22,01

50000 25,25 26,71 0 0 5 10 15 20 25 Time (min)

Figure 4.16a Total ion chromatogram of crude extract CTF9.

I:\DATABASE\...\Imra\Imransajid\CTF9dp1 14.03.2007 21:40:46

RT: 0,00 - 30,00 9,13 9,44 NL: 100 3,81E7 9,56 24,52 25,31 Base Peak 9,07 15,94 23,91 MS CTF9dp1 9,74 15,86 16,77 50 6,92 6,98 14,08 25,40 13,48 5,54 11,16 18,08 3,66 7,19 15,37 18,71 20,54 25,60 28,48 28,95 1,14 3,60 12,67 20,93 23,68 0 9,92 NL: 1,35 5,05E5 8,64 10,27 11,89 13,70 14,53 Tot al Scan 400000 5,40 6,23 8,09 15,83 16,92 PDA 17,45 CTF9dp1 1,63 3,81 4,19 20,57 uAU 200000 18,76 21,55 22,01 25,25 26,71 28,84 29,56 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 Time (min)

CTF9dp1 #691 RT: 15,86 AV: 1 NL: 6,25E6 T: + c ESI sid=10,00 Full ms [ 100,00-2000,00] 959,7 100

60 1746,3 718,5 40 884,2 1034,7 460,9 523,7 610,1 659,4 819,2 883,1 1142,9 20 1235,8 1256,2 1426,1 1497,9 1577,6 1736,9 1765,8 1861,7 Relative Abundance 987,3 1905,0

0 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 m/z

CTF9dp1 #693 RT: 15,91 AV: 1 NL: 4,66E6 T: + c sid=10,00 d Full ms2 959,69@35,00 [ 250,00-1930,00] 941,4 100

40 923,5 20 905,5 Relative Abundance 675,4 775,4 869,4 441,3 541,5 585,4 959,3 1237,4 0 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 m/z

Figure 4.16b Selected ion chromatogram of extract CTF9 (first line), UV chromatogram, mass spectrum of the signal at Rt = 15.86 min, and MS/MS fragmentation (bottom) of the ion signal [M+H] at m/z 959.

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RT: 0,00 - 30,00 28,72 NL: 100 9,89E6 90 Base Peak MS 80 ctf13dn1

50 22,67 40 17,06 RelativeAbundance 30 22,75 20 20,62 22,83 16,93 18,41 25,62 10 25,23 26,01 0,15 3,11 4,23 6,52 8,06 9,08 10,91 14,05 15,93 0 28,65 NL: 350000 3,62E5 22,55 Total Scan PDA 300000 ctf13dn1

250000

200000 10,84 uAU 150000

100000 16,95 20,78 1,39 11,19 50000 8,68 20,52 23,00 7,00 7,26 12,45 24,99 3,50 4,14 0 0 5 10 15 20 25 30 Time (min)

Figure 4.17a Total ion chromatogram of crude extract CTF13.

I:\DATABASE\Neue Daten\Imra\ctf13dn1 06.06.2007 22:28:54

RT: 0,00 - 30,00 28,72 NL: 100 9,89E6 Base Peak MS ctf13dn1 50 22,67 17,06 17,13 18,41 20,62 25,62 0,15 1,17 1,83 3,11 4,23 4,86 6,78 8,06 9,08 10,42 10,91 11,51 13,36 14,05 15,93 25,33 25,81 0 28,65 NL: 22,55 3,62E5 300000 Tot al Scan PDA

200000 10,84 ct f 13dn1 uAU 100000 16,95 20,78 1,39 7,26 8,68 11,19 12,45 18,35 20,52 23,00 1,21 3,50 4,14 6,01 7,00 10,63 14,36 15,83 23,81 25,44 26,64 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Time (min)

ctf13dn1 #957 RT: 25,46 AV: 1 NL: 2,00E4 T: - c sid=10,00 d Full ms2 909,51@35,00 [ 240,00-1830,00] 863,4 100

40 1710,5 20 Relative Abundance 374,6 574,2 621,7 876,3 0 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 m/z

ctf13dn1 #954 RT: 25,36 AV: 1 NL: 1,05E4 T: - c sid=10,00 d Full ms2 909,47@35,00 [ 240,00-1830,00] 863,5 100

20 439,3 621,1 706,3 891,5

Relative Abundance 589,2 810,5 1155,0 0 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 m/z

Figure 4.17b Selected ion chromatogram of extract CTF13 (first line), UV chromatogram, mass spectrum of the signal at Rt = 25.41 min, and MS/MS fragmentation (bottom) of the ion signal [M+H] at m/z 863.

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RT: 0,00 - 30,06 16,78 NL: 100 6,35E6 90 14,39 Base Peak 28,89 MS 80 16,33 ctf15dp1 70 20,76 25,77 60 17,35 14,32 26,81 7,11 25,68 50 20,89 25,50 40 9,82 9,89 10,12

RelativeAbundance 24,31 30 8,19 10,20 21,04 11,96 13,29 20 5,58 17,64 1,03 5,50 23,50 10 1,43 18,80

0 22,50 NL: 28,76 4,81E5 450000 Total Scan 400000 PDA ctf15dp1 350000 22,17 300000

250000 uAU 200000

150000

100000 20,41 25,09 1,39 16,87 50000 7,23 10,25 10,79 14,29 7,00 25,86 3,34 5,60 0 0 5 10 15 20 25 30 Time (min)

Figure 4.18a Total ion chromatogram of crude extract CTF15.

I:\DATABASE\Neue Daten\Imra\ctf15dp1 06.06.2007 23:50:31

RT: 0,00 - 30,06 16,78 NL: 100 6,35E6 14,39 28,89 16,33 Base Peak 16,85 20,76 25,77 MS ctf15dp1 7,11 26,81 50 14,46 25,59 7,18 9,89 10,12 26,97 9,74 20,96 24,31 5,58 11,96 13,29 14,54 17,64 20,63 21,88 1,03 1,43 4,28 5,50 27,44 0 22,50 NL: 28,76 4,81E5 400000 Tot al Scan 22,17 PDA ct f 15dp1

uAU 200000 1,39 7,23 10,25 10,79 16,70 16,87 18,27 20,41 22,99 25,09 1,81 3,34 4,14 5,97 7,00 8,39 12,36 14,29 25,86 27,95 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Time (min)

ctf15dp1 #1036 RT: 28,82 AV: 1 NL: 4,30E6 T: + c ESI sid=10,00 Full ms [ 100,00-2000,00] 377,3 100

20 774,9 Relative Abundance 717,5 822,6 976,0 148,8 301,1 362,4 494,7 579,4 1110,0 1134,4 1297,0 1363,6 1500,4 1528,1 1588,2 1720,3 1769,2 1966,1 0 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z

ctf15dp1 #1043 RT: 29,00 AV: 1 NL: 2,53E6 T: + c sid=10,00 d Full ms2 377,38@35,00 [ 90,00-765,00] 362,4 100

80 377,3 60

20 378,3 Relative Abundance 203,2 240,2 279,9 335,2 361,7 404,6 0 100 150 200 250 300 350 400 450 500 550 600 650 700 750 m/z

Figure 4.18b Selected ion chromatogram of extract CTF15 (first line), UV chromatogram, mass spectrum of the signal at Rt = 28.82 min, and MS/MS fragmentation (bottom) of the ion signal [M+H] at m/z 377.

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Biological and Chemical Screening

RT: 0,00 - 30,00 11,79 NL: 100 1,68E7 11,88 90 Base Peak MS 80 16,14 ctf24dp1 70

40 17,56 RelativeAbundance 30 20,75 18,69 20 9,62 29,59 5,80 12,21 20,88 26,83 5,88 10 5,43 12,37 21,40 24,64 27,01 0,06 4,41 0 1,37 NL: 6,86 9,94E4 90000 Total Scan PDA 80000 ctf24dp1 70000

60000 7,92 10,12

50000 6,11 11,60 20,66 uAU 5,80 9,27 16,61 40000 3,99 13,47 14,43 30000 17,17 21,54 28,98 20000 22,61 24,56

10000 3,19

0 0 5 10 15 20 25 30 Time (min)

Figure 4.19a Total ion chromatogram of crude extract CTF24.

I:\DATABASE\Neue Daten\Imra\ctf24dp1 07.06.2007 07:19:29

RT: 0,00 - 30,00 11,79 NL: 100 11,88 1,68E7 16,14 Base Peak MS ctf24dp1 50 11,72 17,56 12,13 18,69 20,75 5,73 5,80 9,62 9,70 15,97 21,11 26,83 29,59 0,06 1,21 4,01 4,41 6,81 8,27 14,25 22,25 24,64 27,01 0 1,37 NL: 6,86 9,94E4 Tot al Scan 1,21 7,92 PDA 10,12 20,66 ct f 24dp1 50000 5,80 6,11 9,27 11,60 16,61 uAU 3,99 13,47 14,16 15,57 17,17 17,66 28,98 19,24 21,54 22,08 23,26 24,56 26,74 27,11 2,01 3,19 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Time (min)

ctf24dp1 #779 RT: 21,03 AV: 1 NL: 1,40E6 T: + c ESI sid=10,00 Full ms [ 100,00-2000,00] 935,6 100 301,1 80 149,1 204,9 60 936,3 932,8 40 1848,6 394,4 813,4 1691,6 1776,8 20 445,3 989,6 1121,7 1849,7 Relative Abundance 310,8 579,0 718,5 1667,1 1933,6 1245,5 1313,5 1498,4 1548,7 0 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z

ctf24dp1 #780 RT: 21,06 AV: 1 NL: 1,64E5 T: + c sid=10,00 d Full ms2 935,60@35,00 [ 245,00-1885,00] 797,5 100

80 883,5

60 711,3 840,6 1159,5 1055,4 1245,4 40 539,2 625,2 453,2 892,9 987,4 1227,5 367,0 643,3 712,4 1073,4 1331,4 1418,5 20 969,3 Relative Abundance 557,3 1486,6 299,1 385,3 454,3 1571,9 1591,7 0 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 m/z

Figure 4.19b Selected ion chromatogram of extract CTF24 (first line), UV chromatogram, mass spectrum of the signal at Rt = 21.03 min, and MS/MS fragmentation (bottom) of the ion signal [M+H] at m/z 936.

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0 6,98 NL: 160000 1,61E5 1,38 Total Scan 140000 PDA 5,98 ctf25dp1 120000 10,76 8,64 100000 11,10 12,38

80000 13,54 uAU 14,22 60000 16,65 20,61 5,54 17,22 40000 21,45 4,31 22,68 3,28 26,42 20000 28,68

0 0 5 10 15 20 25 30 Time (min)

Figure 4.20a Total ion chromatogram of crude extract CTF25.

I:\DATABASE\Neue Daten\Imra\ctf25dp1 07.06.2007 02:33:46

uAU 14,22 16,65 20,61 50000 5,54 17,22 18,27 2,28 3,28 4,31 21,45 21,96 24,34 26,42 28,68 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Time (min)

ctf25dp1 #826 RT: 22,79 AV: 1 NL: 1,50E6 T: + c ESI sid=10,00 Full ms [ 100,00-2000,00] 919,6 100

40 148,9 941,7 279,1 301,2 373,1 717,5 815,4 888,6 20 435,1 611,6 653,7 Relative Abundance 251,1 951,1 1081,6 1277,6 1388,6 1529,0 1161,3 1640,6 1732,6 1867,5 1996,7 0 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z

ctf25dp1 #827 RT: 22,82 AV: 1 NL: 1,33E6 T: + c sid=10,00 d Full ms2 919,57@35,00 [ 240,00-1850,00] 887,6 100

40 855,7

20 Relative Abundance 545,5 612,2 663,3 783,2 837,6 919,0 1208,5 0 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 m/z

Figure 4.20b Selected ion chromatogram of extract CTF25 (first line), UV chromatogram, mass spectrum of the signal at Rt = 22.79 min, and MS/MS fragmentation (bottom) of the ion signal [M+H] at m/z 920.

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Biological and Chemical Screening of strains RSF17, RSF18, RSF23 and BG5 exhibited 8-10 signals at different retention times each corresponding to a particular metabolite, similarly the crude extracts of Strains CRF1, CRF2, CRF11, CRF14 and CRF17 exhibited 5-8 signal at different retention times (Table 4.2). For the whole HPLC MS data, between zero (e.g. in extract CRF11) and more than 100 hits (e.g. SCF18) were found for each signal (Table 4.2). The minimum number of hits in AntiBase was found for the compounds of strains RSF17, RSF18, BG5, CRF11, CRF17 and SCF31. The strains SCF18, CTF9, CTF15 were found to produce large numbers of (more than 10) metabolites (Table 4.2). A missing database entry indicates that the respective compound most likely has not been isolated from microorganisms before, a higher number of hits for a mass is not more than a measure for the probability to re-isolate known metabolites. The total ion chromatograms and one selected ion chromatogram for each of the crude extract of Streptomyces strains analysed by HPLC-MS are given in figures 4.4a – 4.20b.

Discussion

The methods employed in this scheme of prescreening provided deeper insights into the metabolic diversity and antimicrobial activities of the Streptomyces strain collection. Biological screening results proved the strains as potent producers of bioactive secondary metabolites and clearly suggested them as a good source of interesting antibacterial, antifungal and anti-microalgal compounds. It has been estimated that the genus Streptomyces might produce at least 100,000 new compounds of biological interest (Watve et al. 2001). During screening of new isolates for drug discovery, many potentially interesting microorganisms might be excluded due to inadequate techniques, too high selectivities or just because of missing tests (Taddei et al. 2006). Hence it was decided to use a so-called horizontal screening, i.e. a combination of tests with low selectivity covering a broad range of antibacterial, antifungal, phycotoxic and cytotoxic activities (Laatsch 2000). Additionally we utilized the chemical screening approach to establish similarities or differences between the secondary metabolite patterns of our isolates assuming that each strain produces a specific metabolic fingerprint when it is grown under the same culture conditions. Chemical screening by TLC using selected

113

Biological and Chemical Screening spraying reagents and HPLC-MS analysis showed a pattern of secondary metabolites specific for each strain and provided deep insight into the metabolites of a strain ensuring its qualification or disqualification for further work. In case of thin layer chromatography

(TLC, CH2Cl2/5% MeOH as solvent system), anisaldehyde/H2SO4 as spray reagent gave the best results, due to its capability to stain the various spots with different colors. The characteristically colored bands produced on TLC plates were marked and documented by scanning (Figure 4.3). HPLC-MS analysis of the crude extracts produced by these strains gave an additional picture of their metabolic diversity (Table 4.2). The molecular masses resulting from HPLC-MS/MS measurements of the crude extracts were searched in AntiBase, which is a database for rapid structure determination of microbial natural products (Laatsch, AntiBase 2007): A hit number >0 in AntiBase reflects a high probability that the compound may already be known; in contrast, a hit number of 0

(occurred only once, for Rt 18.53 in CRF11) indicates clearly that the compound in question has probably not been isolated from micro-organisms before. The highest molecular weight of m/z 1652 Dalton was measured for a constituent in the crude extract of strain CTF24 at retention time Rt=20.65 min. The AntiBase search in a range of 1 amu gave four hits for this mass, among them the glycopeptide phleomycin G, and a microviridin. Also other high molecular weight compounds with the masses of m/z 1330, 1186, 1162, 1131, 1127, 1115, 1035, 1022 and 1007 Dalton, observed in the crude extracts of strains CRF11, SCF31, RSF17, CRF17, RSF18, and CTF23 respectively, resulted mainly in microbial peptides in AntiBase seach. HPLC-MS results from the crude extracts of isolates SCF18, CTF9 and CTF15 showed that these strains have the ability to produce a large number of metabolites in parallel. The bioautography results of the extracts of strain CRF17, CRF14 exhibited that most of the components in these extracts are bioactive except one or two fractions close to the solvent front of the chromatogram that might be lipids or fats, while in the extract of RSF18 only on fraction exhibited activity (Figure 4.2).

As HPLC-MS alone did not provide satisfactory information for a complete dereplication (= rapid identification of known compounds), so additionally tandem mass spectrometry (MS/MS) was applied, i.e. the quasimolecular ions were bombarded with helium atoms at a collision energy of 35% of the instrument's maximum. The fragmentation pattern

114

Biological and Chemical Screening especially of peptides is highly structure-sensitive and allows determining the amino acid sequence often quite easily. For the rapid identification of trivial compounds, the resulting fragment pattern was compared with an MS/MS-database of about 600 frequently occurring microbial metabolites (Yao, 2008). In this way, e.g. diketopiperazines, constituents of the nutrient broth (e.g. daidzein, genistein) or contaminants (e.g. phthalic esters) can be easily identified. In extracts of strain CTF15, cis-cyclo(prolylvalyl) and cis-cyclo(prolylleucyl) were easily identified, other compounds with similarity index between 50 and 70% were tetracenomycin D, isoxanthohumol, resistoflavine, versicolorin C, resistomycin, lagumycin A, and diazaquinomycin. Indeed, the preparative separation delivered resistomycin and tetracenomycin D. Further dereplication is possible by comparison of UV absorptions or the empirical formula (obtainable by HRMS) of the respective signal with data collections (e.g. AntiBase), or by selective chemical reactions: if developed chromatograms of the crude extracts are moistened with 2N NaOH, a color change of yellow or orange spots to blue or violet indicates the presence of peri-hydroxyquinones, as it was the case for strains SCF25, CRF11, CRF14, and BG5. Other chemical reactions are indicative for peptides, prodigiosins, alkaloids etc.

Using this approach of chemical screening including TLC, HPLC MS/MS and database searches, the enormous diversity of metabolites of the isolates was established. As a consequence of the detailed analysis of the results obtained from the biological and chemical screening project it seems reasonable to comment on the secondary metabolism of the analyzed strains and on the whole Streptomyces strain collection (110 strains) in a more general manner. Depending on their metabolic fingerprint visualized by the chemical screening and results of biological activities the interesting strains or the talented organisms were selected for the further work of fermentation, extraction, purification and structure elucidation of the bioactive compounds.

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Fermentation, isolation, purification and structure elucidation

CHAPTER 5

FERMENTATION, ISOLATION, PURIFICATION AND STRUCTURE ELUCIDATION OF THE METABOLITES

The identification of active compounds from fermentation samples (extracts, whole broths) is one of the most complicated, labour intensive and time consuming step of the screening protocols (Berdy, 2005). The great progress in the automated methods of chromatographic isolation and spectroscopic identification techniques (LC-MS, LC-NMR, HPLC-UV-VIS) are more or less satisfying the requirements of the modern screening programs (Hill et al., 1998). In the last decades using MS and NMR methods it became possible to solve the structure in minimum quantities of natural products. The structure determination of a new compound, isolated perhaps only on milligram scale is now a relatively easy task. The adequate quantities of compounds are critical, for detailed pharmacological and clinical tests however the scale up and the production development is usually a simple task. In this study six Streptomyces strains RSF18, CRF14, BG5, CTF9, CTF15 and SCF25 were selected on the basis of pre-screening results. These strains exhibited interesting antimicrobial activities and an impressive chemical diversity of the metabolites in biological and chemical screening. The decision for the selection of these strains for preparative screening was also made on the basis of origin of the strain, from each group of the Streptomyces strain collection one or two strains were selected and the bioactive metabolites produced by them were identified. The strains RSF18, BG5 and CTF15 were cultivated as shaking cultures on a linear shaker, while the strains CRF14, CTF9 and SCF25 were cultivated in lab scale (20-50 L) fermenters. The resulting culture broth of each strain was filtered by a filter press to separate the cell mass from the liquid phase, the cell mass was extracted by ethyl acetate and acetone while the liquid phase was adsorbed on Amberlite XAD-2 resin and the XAD resin was eluted with methanol. The crude extracts obtained were subjected to chromatographic separation and purification techniques (silica gel, preparative TLC and gel exclusion chromatography etc) and the active metabolites were purified. The structure elucidation of the pure fractions was performed by mass spectrometry (MS), NMR spectroscopy and data base searches. From all the selected strains 15 compounds were identified, among them one is the new cyclic thiopeptide derivative while the others are already reported compounds. The compounds obtained from

116

Fermentation, isolation, purification and structure elucidation these Streptomyces strains belong to different structural classes including peptides, macrolides, polyethers and quinones etc. In the following section each of the selected strain has been reported individually along with the strategy adopted for fermentation, extraction, isolation, purification and structure elucidation of the bioactive compounds produced by it.

Streptomyces sp. RSF18 Fermentation and Isolation The strain RSF18 isolated from rose field was inoculated from well grown plates in 60 1L erlenmeyer flasks, each containing 250 ml of GYM medium (Table 2.5). The fermentation was carried out at 95 rpm on the linear shaker for 7 days at 28o C, it formed brown-yellow culture broth, which was filtered over celite and adsorbed on Amberlite XAD-2, while the mycelium was extracted with acetone. TLC-directed work-up of the mycelial extract (6.85 g) by silica gel column chromatography and size exclusion chromatography resulted in two pure compounds (1; 78.6 mg, 2; 27.5 mg). However, the water extract (1.3 g) delivered one pure compound as white solid (3; 19.8 mg). Figure 5.1 summarizes the strategy adopted for the fermentation, extraction, isolation and purification of the active metabolites from the strain RSF18.

The Compounds 1 and 2 were obtained as white amorphous solid with UV absorbing middle polar substances, which turned yellow on TLC by spraying with anisaldehyde/sulphuric acid reagent and were identified using NMR, HRESI MS and MS- MS technique as thiopeptide antibiotics; the geninthiocin (1) and the new val-geninthiocin (2) which is a deoxy-derivative of 1. The physico-chemical properties of compounds 1 and 2 are given in Table 5.1, both are soluble in DMSO, methanol, ethanol and ethyle acetate but are insoluble in hexane and dichloromethane. The 1H and 13C NMR spectral data of geninthiocin (1) and val-geninthiocin (2) are summarized in Table 5.2. The 13C NMR spectrum of val-geninthiocin (2) revealed the presence of same carbon numbers as in the known geninthiocin (1) i.e. 50 carbons. While its 1H NMR spectrum showed 49 protons including only 12 exchangeable ones, in geninthiocin (1) it showed 13 exchangeable ones, due to the lose of one of the acidic protons from compound 1 and that it could be one of the OH groups, that is further confirmed from the MS and HRESI MS with less oxygen atom difference from compound 1.

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Fermentation, isolation, purification and structure elucidation

The 1H NMR spectrum of compound 2 showed the same pattern as of 1 (Table 5.2) except the dissapperance of the acidic proton at 5.17. In addition, the replacement of the singlet methyls in compound 1 at 1.22 and 1.20 with doublet signal of 6 H at 0.97 (d, 6.6 Hz) of the iso-propyl group [CH(CH3)2], due to the loss of OH in the hydroxyl-valine in compound 1. This is further confirmed from the 13C NMR spectra of compound 2, which also showed the same pattern of 1 except; the oxy-carbon at 71.0 dissappeared in compound 2 and is replaced with methine group at 29.5, confirming compound 2 as derivative of geninthiocin (1). Of the thiopeptide antibiotics, thioxamycin (Matsumoto et al., 1989), berninamycin (Abe et al., 1988), sulfomycin I (Debono et al., 1992) possess a substructure consisting of thiazole- pyridine-oxazole in the cyclic peptide core, while thiocillin I, micrococcin P (Bycroft and Gowland, 1978) possess a thiazole-pyridine-thiazole moiety, which was named as sulfomycinamate in sulfomycin I. Two adjacent aromatic doublet protons at 8.50 and 8.23 in geninthiocin (1) (J= 8 Hz) and val-geninthiocin (2) by comparison of the chemical shifts with those of the corresponding pyridine moiety in sulfomycin I revealed the presence of a 2,3,6-trisubstituted pyridine. The 13C chemical shifts of the pyridine moiety in sulfomycin I were 149.0, 130.5, 140.0, 121.5 and 146.6 ppm for C-2, C-3, C-4, C-5 and C-6, respectively, which were in good agreement with those of the corresponding carbons in geninthiocin (1) and val-geninthiocin (2) (Table 5.2). This result unambiguously revealed the presence of a thiazole-pyridine-oxazole moiety in the cyclic peptide core as seen in thioxamycin, berninamycin A, sulfomycin I. CID-MS/MS studies Tandem mass spectrometry is a very powerful method for sequence analysis of peptides and proteins. The fragmentation of linear peptides was described comprehensively and detailed knowledge of the fragmentation mechanism has been obtained (Roepstorff and Fohlmann, 1984; Harrison and Yalcin, 1997; Kintner and Sherman, 2000; Piazs and Suhai, 2005). However, the structure analysis of cyclic peptides, which represent an important class of bioactive natural products, by mass spectrometry remains a challenging task. Complex fragmentation patterns by two-bond cleavage at different ring positions, ring-opening reactions and uncommon rearrangement. reactions complicate the interpretation of CID-MS/MS spectra (Ngoka and gross, 1999; Williams and Brodbelt, 2004; Schafer et al., 2006), and frequently

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Fermentation, isolation, purification and structure elucidation

RSF18 (15 l Shaker)

7 days

mixing with cilite and filtered by filterpress

Biomass Filtrate

4 x with EtOAC and XAD-2 (MeOH/H2O), i.vac 1x with acetone i.vac EtOAC

Crude extract Crude extract (6.85 g) (1.3 g)

chrom. on silica gel (CH Cl -MeOH) 2 2 chrom. on silica gel (CH2Cl2-MeOH)

B2, A1 Fat; 3.3 g A2, B1, A1 Fat Chalcomycin; (19.8 mg)

A1 = Sephadex LH-20 (CH2Cl2/40% MeOH)

A2 = Sephadex LH-20 (CH2Cl2/50%MeOH)

Geninthiocin (1); 78.6 mg Val-Geninthiocin; 27.5 mg B1 = PTLC (CH2Cl2/6 % MeOH) B2 = PTLC (CH Cl /5 % MeOH) 2 2 Figure 5.1 Working up scheme of the Streptomyces sp. RSF18

Table 5.1 Physico-chemical properties of geninthiocin (1) and val-geninthiocin (2) Geninthiocin (1) Val-Geninthiocin (2) Appearance White amorphous solid White amorphous solid

Rf 0.55 (CH2Cl2/10% MeOH) 0.61 (CH2Cl2/10% MeOH) Solubility Soluble in DMSO, MeOH, EtOH and EtOAc. Soluble in DMSO, MeOH, EtOH and EtOAc.

Insoluble in hexane, CH2Cl2 Insoluble in hexane, CH2Cl2.

Molecular formula C50H49N15O15S C50H49N15O14S (+)-ESI-MS: m/z (%) 1154.3 [M+Na]+ 1138.4 [M+Na]+ (-)-ESI-MS: m/z (%) 1130.2 [M-H]- - (+)-HRESI-MS: 1132.3331930 [M+H]+ and 1154.31444 [M+Na]+ 1116.3370680 [M+H]+ and 1138.31812 [M+Na]+ Found

Calc. 1132.332595 [M+H], for C50H50N15O15S and 1116.337675 [M+H], for C50H50N15O14S and

1154.314535 [M+Na], for C50H50N15O15S Na 1138.319625 [M+Na], for C50H49N15O14S Na

UV/VIS: max (log ) (MeOH): 237 (5.09); (MeOH/HCl): 238 (5.04); (MeOH): 237 (4.93); (MeOH/HCl): 239 (4.92), (MeOH/NaOH): 238 (5.04) nm. (0.20 mg/10 ml MeOH) 205 (4.94); (MeOH/NaOH): 239 (4.92) nm.

25 [ ] D (1 mg/1 ml MeOH) + 155° + 131°

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Fermentation, isolation, purification and structure elucidation

Table 5.2 13C and 1H NMR of geninthiocin (1) and val-geninthiocin (2) compared with literature in DMSO-d6.

Position Geninthiocin (1) (Lit. data) Geninthiocin (1) Exp. Val-Geninthiocin (2)

C H (J in Hz) C H C H Thiazole C-2 163.2 - 163.1 - 163.0 - C-4 149.4 - 149.4 - 149.4 - CH-5 126.9 8.94 126.7 8.70 (s) 126.6 8.70 CO 159.9 - 159.9 3.51 (d, 7.2) 159.8 - Threonine NH - 8.00 (d, 9.0) - 8.00 (d, 9.0) 8.00 (d, 9.2) CH 57.8 4.60 (dd, 9.0, 3.0) 57.7 4.60 (dd, 9.0, 3.0) 57.6 4.60 (dd, 9.0, 3.0) CH 67.3 4.29 (m) 67.2 4.29 (m) 67.3 4.29 (m) CH3 20.5 1.14 (d, 6.2) 20.4 1.14 (d, 6.2) 20.3 1.14 (d, 6.1) OH - 4.98 (d, 5.5) - 4.98 (d, 5.5) - 4.95 (d, 5.4) CO 168.8 - 168.7 - 168.6 Oxazole (1) NH - 9.60 - 9.55 (s) - 9.60 (s br) C 123.1 - 123.1 - 123.0 - CH 129.5 6.55 (q, 7.3 ) 129.5 6.55 (q, 7.3 ) 129.2 6.53 (q, 7.0 ) CH3 13.8 1.74 (d, 7.3) 13.7 1.74 (d, 7.3) 13.6 1.75 (d, 7.0) C-2 159.4 - 159.4 - 159.4 - C-4 136.1 - 136.0 - 136.1 - CH-5 142.7 8.70 142.6 8.70 (s) 142.5 8.70 (s) CO 158.4 - 158.3 - 158.3 - Dehydroalanine (1) NH - 9.39 - 9.39 (s) - 9.40 (s br) C 133.4 - 133.4 - 133.3 - CH2 103.7 6.46, 5.88 103.7 6.46, 5.88 105.8 6.46 (s), 5.88 (s) CO 163.7 - 163.7 - 163.7 - Hydrovaline and NH - 8.23 (d, 8.5) 8.23 (d, 8.5) 8.25 (d, 8.1) valine CH 61.8 4.64 (d, 8.5) 61.7 4.64 (d, 8.5) 60.0 4.61 (dd, 8.8, 2.8) C 71.0 - 71.0 - 29.5 2.20 (m) CH3 27.3 1.22 27.3 1.22 (s) 18.9 0.97 (d, 6.6) CH3 26.1 1.20 26.0 1.20 (s) 18.8 0.97 (d, 6.6) OH - 5.17 - 5.17 (s) - - CO 169.4 - 169.3 - 171.0 - Oxazole (3) NH - 9.61 - 9.60 - 9.60 C 128.5 - 128.5 - 128.6 - CH2 105.6 6.13, 5.65 105.7 6.13, 5.65 106.1 6.09, 5.70 C-2 155.2 - 155.1 - 155.1 - C-4 129.2 - 129.2 - 129.2 - C-5 154.6 - 154.5 - 154.2 - CH3-5 11.5 2.62 11.5 2.62 11.4 2.63 (s) CO 159.5 - 159.5 - 159.4 - Dehydroalanine (2) NH - 9.36 - 9.36 - 9.37 (s br) C 133.9 133.9 133.9 CH2 105.9 6.36, 5.76 105.9 6.36, 5.76 105.8 6.36, 5.76 CO 162.7 162.7 162.6 Oxazole (2) NH - 9.77 - 9.77 - 9.69 C 129.3 129.1 130.0 CH2 111.2 5.70, 5.71 111.0 5.70, 5.71 111.2 5.70, 5.73 C-2 158.1 - 158.3 - 158.0 - C-4 139.2 - 139.2 - 139.1 - CH-5 140.5 8.68 140.5 8.68 140.4 8.68 (s) Pyridine C-2 149.2 - 149.2 - 149.2 - C-3 130.2 - 130.1 - 130.1 - CH-4 141.0 8.50 (d, 8.0) 141.0 8.50 (d, 8.0) 140.8 8.52 (d, 8.2) CH-5 121.5 8.23 (d, 8.0) 121.3 8.23 (d, 8.0) 121.4 8.25 (d, 8.1) C-6 146.8 - 146.8 - 146.8 - CO 161.6 - 161.6 - 161.5 - Dehydroalanine (3) NH - 10.53 - 10.53 - 10.51 (s br) C 134.7 - 134.7 - 134.7 - CH2 106.2 6.42, 5.83 106.1 6.42, 5.83 106.0* 6.43, 5.84 CO 162.0 - 162.0 162.0 Dehydroalanine (4) NH - 9.44 - 9.44 - 9.42 (s br) C 135.1 - 135.0 - 135.0 - CH2 106.0 6.03, 5.83 106.0 6.03, 5.83 106.0 6.05, 5.70 CO 165.1 - 165.1 - 165.0 - NH2 - 7.93, 7.50 - 7.93, 7.50 - 7.92, 7.48

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Figure 5.2 1H NMR (DMSO, 300 MHz) of genenthiocin (1)

Figure 5.3 1H NMR (DMSO, 300 MHz) of val-genenthiocin (2)

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Fermentation, isolation, purification and structure elucidation

Figure 5.4 13C NMR (DMSO, 125 MHz) of genenthiocin (1).

Figure 5.5 13C NMR (DMSO, 125 MHz) of val-genenthiocin (2). Deala (3) O CH O 2 H Pyr N N NH H 2 N Deala (4) O CH2 Thz S O Oxa (2) N N Thr O O NH CH3 CH2 CH NH 3 HN HO O Deala (2) H2C Oxa (1) O N NH

O H C H 3 CH3 N O N O R CH H 3 N O H2C N Oxa (3) H CH Deala (1) O 2 Hyval and val 1: Geninthiocin R = OH 2: Val-Geninthiocin R = H Figure 5.6 Molecular structure of the cyclic thiopeptides geninthiocin and val-geninthiocin

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Fermentation, isolation, purification and structure elucidation

higher order MSn investigations are required. Furthermore, cyclic peptides produced as secondary metabolites by bacteria contain uncommon amino acid residues leading to different fragmentation reactions. It was therefore of interest to investigate the fragmentation behavior of compounds 1 and 2 and to further confirm the NMR-derived structure of compound 2 by MS investigations. For this purpose, MS2 and MS3 experiments using a quadrupol ion trap were performed (Table 5.3), and additionally, high- resolution CID-MS/MS measurements were carried out on a FT-ICR mass spectrometer to determine the elemental composition of key fragments (Table 5.4). To compare the fragmentation behavior of geninthiocin (1) and val-geninthiocin (2), CID-MS/MS investigations were carried out on the [M+H]+, [M+Na]+, [M+2H]2+ and [M-H]- ions. In general, for sequencing linear peptides, fragmentation of the [M+2H]2+ ions is used successfully, but in the case of the geninthiocin (1) and val-geninthiocin (2), fragmentation spectra of those ions were dominated by doubly charged fragment ions without significant sequence information. Furthermore, [M+2H]2+ ions were observed with very low intensity under the conditions described. Also the MS2 spectra of [M+Na]+ and [M-H]- provided little sequence information. Therefore, CID-MS/MS studies were concentrated on the more abundant [M+H]+ species (Fig 5.7). For the fragmentation mechanism, the position of the proton is essential. It can be proposed that the proton of the [M+H]+ precursor ion is placed on the terminal amide N of the side chain and fragmentation occurs by cleavage of neutral amino acid residues from the ring system and from the side chain.

Beside an unspecific loss of H2O, CO, and NH3, the following main fragmentation pathways were observed (Latin numbers correspond to the fragments indicated in Figure 5.8): 1. Cleavage of a single amino acid residue unit from the peptide ring system is found basically for Hyval (-115 Dalton) in 1 and Val (-99 Dalton) respectively for 2 (cleavage of two CO-NH peptide bonds). Therefore, the structural difference between both compounds can be located at this amino acid at a very early stage.

2. Cleavage of a single amino acid residue unit together with NH3 from the peptide

ring system (H2N-Oxa-NH2, H2N-Hyval/Val-NH2, H2N-Deala-NH2 (cleavage of

one CO-NH peptide bond and one CCH2-NH bond) as well as from the side chain

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Fermentation, isolation, purification and structure elucidation

(H2N-Deala(4)-NH2, b-fragment ion) were detected. Obviously, the -NH-CCH2- bond between Oxa(1)-Deala(1) and Oxa(3)-Deala(2) as well as between Deala(2)- Oxa(2) and Hyval/Val-Oxa(3) can be broken favorably leading to a second preferred fragmentation pathway beside peptide bond (-CO-NH-) cleavage. 3. Cleavage of dipeptide units Oxa-Deala with high intensity resulting in the prominent fragment ion peaks at m/z 913 and 897 respectively. Furthermore, fragmentation of Deala-Hyval/Val occurs with lower intensity. 4. Cleavage of the tripeptide unit Hyval/Val-Oxa-Deala gives the key fragment at m/z 798. 5. Cleavage of the tripeptide unit Hyval/Val-Oxa-Deala followed by a further dipeptide Oxa-Deala loss results in the fragment ion m/z 579. 6. Cleavage of a dipeptide unit Deala(3)-Deala(4) from the side chain by

fragmentation of the NH-CCH2 bond between Deala(3) and Pyr (c-fragment ion). These fragmentation pathways derived from collision activated dissociation using a quadrupole ion trap were confirmed by high resolution MS/MS in a FT-ICR mass spectrometer providing the elemental composition of key fragments. Table 5.4 shows the results of the exact mass determination and the calculated elemental composition. In summary, electrospray tandem mass spectrometry (MS2 and MS3) provided partial sequence information of both geninthiocins. By comparison of MS2 spectra, the position of valine in compound 2 was determined unambiguously and the NMR-derived structure was confirmed. The obtained information on fragmentation pathways can be applied on further investigations on this class of substances to identify low amounts of derivatives.

From the water phase extract, compound 3 was isolated as white solid after subjecting to

PTLC (CH2Cl2/5%MeOH) and further purified on Sephadex LH-20 (CH2Cl2/40%MeOH). The 1H-NMR spectrum (Fig 5.9) showed in the olefinic region a 1H dd signal at =6.66 (3J=15.4 and 10.2 Hz), one 2H-multiplet at =6.58 and one doublet at =5.82 (3J=15.4 Hz). A further quartet of a doublet with intensity 1 at =5.34, two 1H doublets at =4.58 and 4.22, many 1H multiplets typical for sugar residues between =4.0 and 3.0 and three methoxy signals at =3.63, 3.57 and 3.42 were identified. Additionally, the spectrum

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Fermentation, isolation, purification and structure elucidation

897 100 95 90 1098 85 80 949 75 70

60 55 50 45 1088 1099 798 Relative Abundance 40 1070 35 30 1017 25 978 1030 20 852 879 631 931 15 780 10 508 579 746 613 850 5 370 660 729 465 492 545 1116 0 400 500 600 700 800 900 1000 1100 1200 m/z Figure 5.7 CID-MS/MS spectrum of val-geninthiocin (2); fragmentation of [M+H]+ measured on a quadrupole ion trap instrument

VI c 9 VI b10

O CH O 2 H Pyr N N NH H 2 N Deala (4) O CH2 Thz S Deala (3) O Oxa (2) N N Thr O O NH CH3 CH2 CH NH 3 HN Deala (2) HO O H2C Oxa (1) NH III O N Hyval and val Deala (1) O H C H 3 CH3 N O N O R CH II H 3 N O H C N Oxa (3) 2 H CH O 2

I Figure 5.8 Selected CID-MS/Ms key fragments of the [M+H]+ precursor ions of geninthiocin (1, R = OH) and Val-geninthiocin (2, R = H) obtained on a quadrupole ion trap mass spectrometer

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Table 5.3 MS2 and MS3 product ions of the [M+H]+ precursor ions of geninthiocin (1) and val-geninthiocin (2) obtained on a quadrupol ion trap mass spectrometer.

Val- Geninthiocin Geninthiocin Fragment ions Main fragment Fragment ions Fragments MS3 Proposed MS2 m/z ions MS3 m/z MS2 m/z m/z Fragmention MS2 1115 1099 -NH3 1114 1098 -H2O 1104 1088 -CO 1056; 1046; 988; 1074 936; 907; 855; -C H O from Hyval 3 6 812; 798 1028; 1018; 985; 1046 1030 1012; 1002 -(Deala + NH ) 949; 879; 851 3 1000; 999; 982; 1000; 999; 931; 1017 931; 879; 862; 1017 -Hyval/Val 879; 798; 579; 493 798; 579 -C H N O from side 994 966; 936; 974; 370 978 960; 950; 370 6 6 2 2 chain 948; 947; 921; 931; 921; 905; 949 965 907; 903; 879; 903; 863; 850; - (Oxa + NH ) 3 850; 827; 798; 631 811; 631 947 931 -(Oxa + NH3) - H2O 896; 895; 877; 897 879; 851; 833; 913 -Oxa-Deala 855; 852; 837; 798 811; 798; 793; 579 907 889; 879; 863; -C H N O 10 15 3 3 821; 769; 752; 631 -(Oxa + NH3) - C3H6O 895 878; 877; 851 879 861; 851 -Oxa – Deala - H2O -C H N O m/z 907 889 10 17 3 4 –H2O -Hyval/Val - (Oxa + 850 850 NH3) 781; 780; 770; 781; 780; 770; -Hyval/Val – Oxa - 798 754; 712; 660; 798 754; 712; 660; Deala 631; 579 631; 579 -Hyval/Val -Oxa- 780 780 Deala-H2O -Deala – Oxa - 729; 728; 660; 729; 728; 660; 746 746 Hyval/Val - COCCH 608; 579 (608; 579) 2 + 2H 603; 545; 493 631 -Hyval/Val – Oxa – 631 603; 545; 493 Deala -(Oxa-NH3) 561; 551; 535; -Hyval/Val – Oxa – 579 551; 535; 493; 441 579 493; 441 Deala – Oxa - Deala 508 370 508 370 370 370

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Fermentation, isolation, purification and structure elucidation

Table 5.4 Confirmation of key fragments by exact mass determination using a FT-ICR mass spectrometer. Geninthiocins (1) Mass (measured) Composition Mass (calculated) m [ppm] Fragmentation + 965.2619 C43H41N12O13S1 965.2631 1.3 [M+H-C7H9N3O2] + 947.2523 C43H39N12O12S1 947.2526 0.2 [M+H-C7H11N3O3] + 907.2203 C40H35N12O12S1 907.2213 1.1 [M+H-C10H15N3O3] + 889.2102 C40H33N12O11S1 889.2107 0.6 [M+H-C10H17N3O4] + 798.2040 C35H32N11O10S1 798.2049 1.1 [M+H-C15H18N4O5] + 746.2095 C32H32N11O9S1 746.2100 0.7 [M+H-C18H18N4O6] + 631.1348 C28H23N8O8S1 631.1354 0.9 [M+H-C22H27N7O7] Val- Geninthiocin (2) Mass (measured) Composition Mass (calculated) m [ppm] Fragmentation + 949.2678 C43H41N12O12S1 949.2682 0.4 [M+H-C7H9N3O2] + 931.2577 C43H39N12O11S1 931.2576 0.1 [M+H-C7H11N3O3] + 897.2723 C40H41N12O11S1 897.2733 1.1 [M+H-C10H9N3O3] + 798.2036 C35H32N11O10S1 798.2049 1.6 [M+H-C15H18N4O5] + 631.1359 C28H23N8O8S1 631.1354 0.8 [M+H-C22H27N7O6]

Table 5.5 Antibacterial and antifungal activities of val-geninthiocin (1) in comparison with geninthiocin (2) and chalcomycin (conc. in 40µg/ disk).

Tested microorganisms Inhibition zone [mm] Geninthiocin Val- Chalcomy (2) geninthiocin (1) cin Bacillus subtilis 15 13 29 Staphylococcus aureus 15 14 36 Streptomyces viridochromogenes 14 11 37 (Tü 57) Escherichia coli - - - Candida albicans 11 11 11 Mucor miehei (Tü 284) 11 11 11 Chlorella vulgaris - - - Chlorella sorokiniana - - - Scenedesmus subspicatus - - -

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Fermentation, isolation, purification and structure elucidation

1 Figure 5.9 H NMR (CDCl3, 300 MHz) of chalcomycin (3).

13 Figure 5.10 C NMR (CDCl3, 125 MHz) of chalcomycin (3). CH O 3

HO CH3 O OH CH3 Formula: C35H56O14 O Mass: 700.811 g/mol O H3C Name: Chalcomycin O CH3 O CH 3 O CH O O 3

OH O O H C CH 3 3 3 Figure 5.11 Molecular structure of chalcomycin

128

Fermentation, isolation, purification and structure elucidation showed multiplets at =2.72(1H), 2.05 (1H) and 1.91 (2H), one methyl singlet at =1.39 and five methyl doublets at =1.35, 1.28, 1.23, 1.21 and 1.01. The 13C-NMR spectrum (Fig 5.10) indicated a ketone signal at =200.2 which must be conjugated with a double bound because of its chemical shift. A further carbonyl signal at =165.3 represented a carboxylic acid, an ester or an amide. Four olefinic methine signals in the sp2 region at =151.8, 146.5, 124.8 and 120.7 could be interpreted as two double bounds conjugated with carbonyl groups. The methane signals at =103.2 and 100.9 were interpreted as due to two acetal carbon atoms. In addition to these signals, 16 signals of carbon atoms connected to oxygen between =87.8-56.7, CH-signals at =49.5, 41.7 and 34.0, methylene- signals at =37.0 and 36.8 and six methyl signals in the range =27.8- 17.8 were observed. MS and NMR followed by searching in Antibase resulted in chalcomycin (3) (Fig 5.11) which was further confirmed by comparison with the literature data.

Biological activities of geninthiocin (1), val-geninthiocin (2) and chalcomycin (3) were determined using the disk diffusion method. In comparison with 1, compound 2 showed slightly lower antibacterial activities against the Gram positive bacteria, Bacillus subtilis, Staphylococcus aureus and Streptomyces viridochromogenes (Tü 57), and minor antifungal activity against Mucor miehei (Tü 284) and Candida albicans. The compounds however exhibited no activities against the Gram-negative bacteria Escherichia coli and the microalgae, Chlorella vulgaris, Chlorella sorokiniana and Scenedesmus subspicatus (Table 5.5).

Streptomyces sp. CRF 17 Fermentation and Isolation The strain CRF 17 isolated from the soil of corn field was cultivated in a 20 litre fermenter (Biostat E) using GYM medium (Table 2.5). The fermenter was filled with 9 litre water along with appropriate quantity of GYM medium and was closed. The inlet and outlet openings and tubes were closed with stoppers and clamps. The pH electrode port was closed with the metal stopper. The fermenter was autoclaved for 30 minutes at 121o C. Afterwards the air supply, stirring motor and water circulation pumps were switched on. The acid (2N HCl), base (2N NaOH) and antifoam (1% Niax/ 70% ethanol) were filled in

129

Fermentation, isolation, purification and structure elucidation their respective jars and were connected to the system. The pH electrode was sterilised with 70% ethanol and adjusted in the pH electrode port in the lid. The preculture was prepared by inoculating the strain CRF 17 from well grown plates into four 1 litre Erlenmeyer flasks each containing 250 ml of GYM medium.

The flasks were incubated at 28o C on a linear shaker for 4 days. Preculture with the ratio of 10% was used to inoculate the biosystem. The Fermentation was carried out for 5 days at 28o C. After harvesting the dark brown culture broth, it was filtered over celite by filter press to separate the mycelium from water phase. The water phase was adsorbed on Amberlite XAD-2, and the Amberlite XAD-2 resin was eluted with methanol, while the mycelium was extracted with ethyl acetate and acetone. TLC of both extracts showed identity which were then collected to gave 0.71 g crude extract. The crude extract was subjected to column chromatography on silica gel using a DCM-MeOH gradient, which produced two fractions. The first fraction was identified as fat, the second fraction was subjected to further purification, using PTLC and Sephadex LH20 which resulted in compound 4 in pure form (Fig 5.12). A band was found in the fraction II, which afforded 17.1 mg of compound 4 as white amorphous solid with medium polarity after being subjected to column chromatography on Sephadex LH-20 (CH2Cl2/40%MeOH). TLC of this compound showed a dark-green colour with anisaldehyde/sulphuric acid on heating, which changed to dark brown after 2 hours.

1 The H NMR in CDCl3 shows two singlets at =6.01 and 6.46 ppm due to the protons of two hydroxyl functions which disappears when D2O is added. The acidic proton does not appear on the spectrum probably because it participates in an intramolecular hydrogen bond (Fig 5.13). The 13C NMR spectrum of this compound confirms the presence of 48 carbon atoms. The signal at =180.7 is due to carboxyl carbon. Between the =67.7 and 108.4, 15 Signals are present which correspond to the carbons bounded to at least one oxygen atom. The off resonance spectrum shows that signals 30, 38, 41, 42, 43, and 44 are due to the 6 quartenary carbons of the molecule. Signals 42, 43, and 44 probably correspond to three hemiacetalcarbons. Lastly, among the signals which appear between 0 and 46, peaks 2, 6 and 12 are probably double (Fig 5.14).

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Fermentation, isolation, purification and structure elucidation

Strain CRF17 Fermenter (10 L) 5 days

mixing with cilite and filtered by filterpress

Biomass Filtrate XAD-2 (MeOH/H O), 4 x with EtOAC and 2 1x with acetone EtOAC i.vac i.vac

Crude extract (0.71 g)

chrom. on silica gel (CH2Cl2-MeOH)

B1, A1 Fat;(41.75 mg) Alborixin; (17.1 mg)

A1 = Sephadex LH-20 (CH2Cl2/40% MeOH)

B1 = PTLC (CH2Cl2/5 % MeOH)

Figure 5.12 Working up scheme of the Streptomyces sp. CRF 17

1 Figure 5.13 H NMR (CDCl3, 300 MHz) of Alborexin (4)

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Fermentation, isolation, purification and structure elucidation

13 Figure 5.14 C NMR (CDCl3, 125 MHz) of Alborexin (4).

Formula: C48 H84 O14 H O Mass: 885 g/mol O O Name: Alborexin H O H O H O O H

O O O H O H O H O

Figure 5.15 Molecular structure of Alborexin (4)

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Fermentation, isolation, purification and structure elucidation

Mass spectrometry revealed that compound 4 has a molecular weight of 885 g/mol.

Furthermore, the HRESI MS gave the molecular formula of C48 H84 O14. Searching in Antibase with this 1H, 13C NMR and MS spectral data resulted in alborexin (4) (Fig 5.15) which was further confirmed by comparison with the literature data (Delhomme et al.,

1976; Gachon et al., 1976; Chapel et al., 1979; Seto et al., 1979).

Streptomyces sp. BG-5

Fermentation and Isolation

The strain BG-5 isolated from the soil of botanical garden was inoculated from well grown plates with pink to red mycelia in 80 1L erlenmeyer flasks, each containing 250 ml of GYM medium (Table 2.5). The fermentation was carried out at 95 rpm on the linear shaker for 7 days at 28o C. It formed dark-pink culture broth, which was filtered over Celite and adsorbed on Amberlite XAD-2, while the mycelium was extracted with ethyle acetate and acetone. TLC-directed work-up of the mycelial extract (2.60g) by silica gel column chromatography and size exclusion chromatography resulted in compound 5 (28.0 mg) along with a very small pure fraction ISBG5C (1.2 mg). The water extract (1.75g) delivered compound 6 (12.8 mg) along with compound 7 (19.8). Figure 5.16 summarizes the strategy adopted for the fermentation, isolation and purification of the three pure compounds from strain BG5. Compound 5 was found in the sub-fraction II, as yellow solid with medium polarity after being subjected to PTLC (CH2Cl2) followed by column chromatography on Sephadex LH-20 (CH2Cl2/40%MeOH). TLC of this compound showed a red colour when treated with 2N NaOH, as indication for peri-hydroxy quinone and gave no colour change with anisaldehyde/sulphuric acid after heating. 1H and 13C NMR (Figure 5.17-18) as well as Mass spectrometry and searching in Antibase resulted in Ochromycenone (5) (Figure 5.19) which was further confirmed by comparison with authentic sample spectra in our collection. Compound 6 was found in the sub-fraction III, which yielded a pale-yellow solid (12.8 mg) with medium polarity after being subjected to column chromatography on Sephadex LH-20 (CH2Cl2/40%MeOH). TLC of this compound showed no colour change when treated with 2N NaOH, which also gave no colour change with anisaldehyde/sulphuric acid after heating. 1H and 13C NMR (Figure 5.20-21) as well

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Strain BG-5 (20 l Shaker)

7 days

mixing with cilite and filtered by filterpress

Biomass Filtrate

4 x with EtOAC and XAD-2 (MeOH/H2O), i.vac 1x with acetone i.vac EtOAC

Crude extract Crude extract (2.60 g) (1.75 g)

chrom. on silica gel (CH Cl -MeOH) chrom. on silica gel (CH2Cl2-MeOH) 2 2

B2, A1 Fat;74.2 mg Fat;5.1 mg

B1, A1 B2, A2 Ochromycinone ISBG5C Emycin D 1-Acetyl- -carbolin (28.0 mg) (1.2 mg) (12.8 mg) (19.8 mg)

A1 = Sephadex LH-20 (CH 2Cl2/40% MeOH) A2 = Sephadex LH-20 (MeOH)

B1 = PTLC (CH2Cl2)

B2 = PTLC (CH2Cl2/5 % MeOH)

Figure 5.16 Working up scheme of the Streptomyces sp. BG-5

1 Figure 5.17 H NMR (CDCl3, 300 MHz) of Ochromycenone (5).

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13 Figure 5.18 C NMR (CDCl3, 125 MHz) of Ochromycinone (5)

Formula: C19 H14 O4 Mass: 306.312 g/mol O CH3 Name: Ochromycinone O

OH O Figure 5.19 Molecular structure of Ochromycinone (5)

1 Figure 5.20 H NMR (CDCl3, 300 MHz) of Emycin D (6)

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Fermentation, isolation, purification and structure elucidation

13 Figure 5.21 C NMR (CDCl3, 125 MHz) of Emycin D (6)

CH3

Formula: C19 H16 O4 Mass: 308.328 g/mol O Name: Emycin-D

O O

OH Figure 5.22 Molecular structure of Emycin D (6)

N N Formula: C13 H10 N2 O1 H Mass: 210.231 g/mol Name: 1-Acetyl- -carbolin O CH 3

Figure 5.23 Molecular structure of 1-Acetyl- -carbolin (7)

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Fermentation, isolation, purification and structure elucidation as Mass spectrometry and Antibase search resulted in Emycin D (6) (Figure 5.22) which was further confirmed by comparison with literature data. As pale yellow solid (19.8 mg) compound 7 was obtained from same sub-fraction III, with medium polarity after being subjected to PTLC (DCM/5% MeOH) followed by column chromatography on Sephadex

LH-20 (CH2Cl2/40%MeOH). TLC of this compound showed a blue fluorescence under long UV which gave yellowish-green colour with anisaldehyde/sulphuric acid on heating. 1 H NMR in CDCl3 and MS data along with AntiBase search resulted 1-Acetyl- -carbolin (7) (Figure 5.23) which was further confirmed by comparison with literature data.

Streptomyces sp. CTF 9

Fermentation and Isolation

The strain CTF 9 isolated from the soil of cotton field was cultivated in a 50 litre fermenter (Biostat U) using GYM medium (Table 2.5). The fermenter was filled with 27 litre water along with appropriate quantity of GYM medium components and was closed. The medium was sterilised by keeping the fermentor in sterilisation mode for 30 minutes at 121o C. Afterwards the air supply, stirring motor and water circulation pumps were switched on. The acid (2N HCl), base (2N NaOH) and antifoam (1% Niax/ 70% ethanol) were filled in their respective jars and connected to the system. The pH electrode was sterilised with 70% ethanol and adjusted in the pH electrode port in the lid. The preculture was prepared by inoculating the strain CTF 9 from well grown plates into four 1 litre o Erlenmeyer flasks each having 250 ml of M2 medium. The flasks were incubated at 28 C on a linear shaker for 4 days. Preculture with the ratio of 10% was used to inoculate the biosystem. The Fermentation was carried out for 5 days. After harvesting the dark brown culture broth, it was filtered over Celite by filter press to separate the mycelium from water phase. The water phase was adsorbed on Amberlite XAD-2, and the Amberlite XAD-2 resin was eluted with methanol, while the mycelium was extracted with ethyl acetate and acetone. TLC of both extracts showed identity which were then collected to gave 5.64 g crude extract. The crude extract was subjected to column chromatography on silica gel using a DCM-MeOH gradient, which produced two fractions. Fraction I was identified as fat, fraction II was subjected to further purification, using PTLC and Sephadex LH20 which resulted in compound 8 (25.7 mg) and compound 9 (12.8) in pure form along with

137

Fermentation, isolation, purification and structure elucidation

Strain CTF 9 Fermenter (50 L) 5 days

mixing with cilite and filtered by filterpress

Biomass Filtrate XAD-2 (MeOH/H O), 4 x with EtOAC and 2 1x with acetone EtOAC i.vac i.vac

Crude extract (5.64 g)

chrom. on silica gel (CH2Cl2-MeOH)

Fat;(1.85 g)

B1, A2 B1, A2 B1, A2,A1 B2, A1 B2, A1 Phenyl acetic acid ISCTF9B -indole-lactic acid ISCTF9E2 ISCTF9G (25.7 mg) (26.4 mg) (12.8 mg) (16.4 mg) (11.8 mg)

A1 = Sephadex LH-20 (CH2Cl2/40% MeOH) A2 = Sephadex LH-20 (100 % MeOH)

B1 = PTLC (CH2Cl2/5 % MeOH)

B2 = PTLC (CH2Cl2/10% MeOH)

Figure 5.24 Working up scheme of the Streptomyces sp. CTF 9

1 Figure 5.25 H NMR (CDCl3, 300 MHz) of Phenyl acetic acid (8)

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Fermentation, isolation, purification and structure elucidation

13 Figure 5.26 C NMR (CDCl3, 125 MHz) of Phenyl acetic acid (8)

Formula: C8 H8 O2 Mass: 136.147 g/mol Name: Phenyl acetic acid OH

O Figure 5.27 Molecular structure of phenyl acetic acid (8)

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Fermentation, isolation, purification and structure elucidation

1 Figure 5.28 H NMR (CDCl3, 300 MHz) of -indole-lactic acid (9).

Formula: C11 H11 N1 O3 OH Mass: 205.210 g/mol Name: -indole-lactic acid OH

N H

Figure 5.29 Molecular structure of -indole-lactic acid (9)

140

Fermentation, isolation, purification and structure elucidation three more unidentified fractions ISCTF9B, ISCTF9E2 and ISCFT9G (Figure 5.24). In Fraction II compound 8 was isolated (25.7 mg) as white solid with medium polarity after being subjected to (DCM/5% MeOH) followed by Sephadex LH-20 (MeOH). The TLC of compound 8 showed a pink colour with anisaldehyde/sulphuric acid on heating. MS, 1H and 13C NMR (Figure 5.25-26) followed by searching in Antibase confirmed it as phenyl acetic acid (8) (Figure 5.27). As 2nd pink colour reaction with anisaldehyde/sulphuric acid on heating the fraction II, compound 9 was isolated as yellow solid (12.8 mg) after being subjected to PTLC (CH2Cl2/5%MeOH), followed by column chromatography on Sephadex LH-20 (MeOH) and Sephadex LH-20 1 (CH2Cl2/40%MeOH). H NMR (Figure 5.28), MS and searching in Antibase resulted in -indole-lactic acid (9) (Figure 5.29) which was further confirmed by comparison with literature data.

Streptomyces sp. CTF 15

Fermentation and Isolation

The terrestrial Streptomyces sp. CTF 15 isolated from the soil of cotton field was inoculated from well grown plates with dark brown mycelia in 80 1L Erlenmeyer flasks, each containing 250 ml of GYM medium (Table 2.5). The fermentation was carried out at 95 rpm on the linear shaker for 7 days at 28o C. It formed a pinkish culture broth, which was filtered over Celite and adsorbed on Amberlite XAD-2. The mycelium was extracted repeatedly with ethyle acetate and acetone, while the Amberlite XAD-2 resin was eluted with methanol. TLC-directed work-up of the mycelial extract (8.75g) by silica gel column chromatography and size exclusion chromatography resulted in compound 10 (60.0 mg) along with compound 11 (13.5 mg). The water extract (1.75g) delivered compound 12 (30.0 mg). Figure 5.30 summarizes the strategy adopted for the fermentation, isolation and purification of the active metabolites from strain CTF15.

A band was found in the fraction II, which afforded 60 mg of yellow solid with medium polarity after being subjected to PTLC (CH2Cl2/5%MeOH) and column chromatography on Sephadex LH-20 (CH2Cl2/40%MeOH). Compound 10 showed under UV at 366 nm as orange fluorescence which gave no colour change with anisaldehyde/sulphuric acid after heating. The 1H NMR spectrum (Figure 5.31) exhibited three siglets at 14.54, 14.34 and 141

Fermentation, isolation, purification and structure elucidation

Strain CTF-15 (20 l Shaker)

7 days

mixing with cilite and filtered by filterpress

Biomass Filtrate

4 x with EtOAC and XAD-2 (MeOH/H2O), i.vac 1x with acetone i.vac EtOAC

Crude extract Crude extract (8.75 g) (0.85 g)

chrom. on silica gel (CH Cl -MeOH) chrom. on silica gel (CH2Cl2-MeOH) 2 2

B2, A1 Fat;85.0 mg Fat;5.1 mg

B2, A1 B2, A1 Resistomycin Tetramycin Actinomycin D ISCTF15D (60.0 mg) (13.5 mg) (30.0 mg) (6.0 mg)

A1 = Sephadex LH-20 (CH 2Cl2/40% MeOH) A2 = Sephadex LH-20 (MeOH)

B1 = PTLC (CH2Cl2) B2 = PTLC (CH Cl /5 % MeOH) 2 2 Figure 5.30 Working up scheme of the Streptomyces sp. CTF-15

1 Figure 5.31 H NMR (CDCl3/MeOH, 300 MHz) of resistomycin (10).

CH H3C 3 HO O

H3C OH

Formula: C22 H16 O6 Mass: 376.359 g/mol Name: Resistomycin OH OH O Figure 5.32 Molecular structure of resistomycin (10)

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Fermentation, isolation, purification and structure elucidation

14.02 characteristis for chelated acidic protons. Three 1H singlets at 7.25, 7.05 and 6.38were assigned to an electron-rich aromatic system. Further more, an aromatic methyl singlet at 2.90 and singlet of intensity 6 at 1.58 attributed to geminal methyl groups were visible. The ESI mass spectra delivered a [M+Na]+ signal at m/z 399 and an [M-H]- signal at m/z 375, respectively, in positive and negative modes fixing the molecular weight at 376 dalton. A substructure search in AntiBase supported by 1HNMR (Figure 5.31) and MS data led to resistomycin (10) (Figure 5. 32) as possible solution, which was further confirmed by comparison with authentic sample spectra in our collection. Compound 11 was found in sub fraction II, which was obtained as a red powder (13.5 mg) with medium polarity after being subjected to PTLC (CH2Cl2/5%MeOH) and column chromatography on Sephadex LH-20 (CH2Cl2/40%MeOH). TLC of this compound showed light pink colour with dark UV absorbance, which also gave no colour change with anisaldehyde/sulphuric acid after heating, however it gave blue colour when treated with NaOH as an indication to hydroxy quinine moity. 1H NMR (Figure 5.33), Mass spectrometry and Antibase search confirmed it as tetracenomycin D (11) (Figure 5.34) which was further confirmed through comparison with literature data.

As an orange-red solid (30 mg) compound 12 was obtained from sub fraction II of liquid phase extract, with medium polarity after being subjected to PTLC (CH2Cl2/5%MeOH) and column chromatography on Sephadex LH-20 (CH2Cl2/40%MeOH). This compound showed light pink fluorescence under long UV, and gave red colour on TLC, while no change in colour with anisaldehyde/sulphuric acid on heating. 1H NMR spectrum(Figure 5.35) of compound 12 showed broad doublets at 8.13, 7.97 and 7.80 of three amide protons , two ortho coupled protons at 7.65 and 7.37 of a 1,2,3,4- tetrasubstituted aromatic ring and two 3H singlets at 2.57 and 2.23 of methyl groups in peri- position of an aromatic system. This is characteristic for the phenoxazinone chromophore in actinomycins. In addition, the spectrum showed overlapping NMR signals at 7.20- 0.75 as it is indicative for a complex peptide. One broad 1H doublet at 7.20(3J = 6.4 Hz), 8 hydrogen signals of oxygenated or -amino acid protons were observed at 6.03 (d), 5.96 (d), 5.25-5.15(m, 2H), 4.78 (d), 4.73 (d), 4.61 (dd) and 4.49 (dd). Unresolved signals of methylene and methane groups with an intensity of further 8H were observed between

143

Fermentation, isolation, purification and structure elucidation

1 Figure 5.33 H NMR (CDCl3/MeOH, 300 MHz) of tetracenomycin (11).

Formula: C19 H12 O6 Mass: 336.295 g/mol Name: Tetracenomycin D

CH3 OH O OH

HO OH

O Figure 5.34 Molecular structure of Tetracenomycin D (11)

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Fermentation, isolation, purification and structure elucidation

Figure 5.35 1H NMR (MeOH, 300 MHz) of actinomycin D (12)

O O N N Formula: C62 H86 N12 O16 O O Mass: 1255.417 g/mol Name: Actinomycin D N N

O O O O

N N O O N N OO

N N O O N N

O O

Figure 5.36 Molecular structure of Actinomycin D (12)

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Fermentation, isolation, purification and structure elucidation

4.03-3.31. Additionally, four singlets with intensity of 12H of four methyl groups were found between 2.93- 2.88 and may be due to N-methyl groups, multiplets of several methylene groups of rings with intensity of 6H appear between 2.65-2.57, multiplets between 2.38-1.76 of 8H and 1.28 (m) of 6H as of two methyl groups. In addition multiplets between 1.17-1.07 with intensity of two methyl groups and 0.99-0.85 for 12H as for four methyl and finally, a doublet of two methyl groups at 0.75(3J = 4.9 Hz) were exhibited.

1H NMR and Mass spectrometry along with Antibase search resulted in Actinomycin D (12) (Figure 5.36) which was further confirmed by comparison with the literature data.

Streptomyces sp. SCF-25

Fermentation and Isolation

The strain SCF-25 isolated from the soil of sugar cane field was cultivated in a 50 litre fermenter (Biostat U) using GYM medium (Table 2.5). The fermenter was filled with 27 litre water along with appropriate quantity of GYM medium and was closed. The medium was sterilized by keeping the fermenter in sterilization mode for 30 minutes at 121o C. The acid (2N HCl), base (2N NaOH) and antifoam (1% Niax/ 70% ethanol) were filled in their respective jars and connected to the system. The preculture was prepared by inoculating the strain SCF 25 from well grown plates into six 1 litre erlenmeyer flasks each having 250 ml of GYM medium. The flasks were incubated at 28o C on a linear shaker for 4 days. Preculture with the ratio of 5% was used to inoculate the biosystem. The Fermentation was carried out for 5 days at 28o C. After harvesting the yellowish culture broth, it was filtered over Celite by filter press to separate the mycelium from water phase. The water phase was adsorbed on Amberlite XAD-2, and the Amberlite XAD-2 resin was eluted with methanol, while the mycelium was extracted with ethyl acetate and acetone. TLC of both the extracts showed similarity which were later mixed to give (2.35 g) of crude extract collectively. The crude extract was subjected to column chromatography on silica gel using a DCM-MeOH gradient, which produced two fractions, fraction I was identified as fat, while working up of the fraction II with preparative TLC and gel exclusion chromatography yielded compound 13 (11.8 mg), along with an unidentified fraction ISSCF25D (10.7 mg) and compounds 14 and

146

Fermentation, isolation, purification and structure elucidation

Strain SCF 25 Fermenter (50 L)

5 days

mixing with cilite and filtered by filterpress

Biomass Filtrate XAD-2 (MeOH/H O), 4 x with EtOAC and 2 1x with acetone EtOAC i.vac i.vac

Crude extract (2.35 g)

chrom. on silica gel (CH2Cl2-MeOH)

B1, A1 Fat;(0.75 g)

B1, A1 B1, A2 B1, A1

Ferulic acid and Tyrosol Tryptophol ISSCF25D (25.4 mg) (11.8 mg) (10.7 mg)

A1 = Sephadex LH-20 (CH2Cl2/40% MeOH) A2 = Sephadex LH-20 (MeOH)

B1 = PTLC (CH2Cl2/5 % MeOH)

Figure 5.37 Working up scheme of the Streptomyces sp. SCF 25

Figure 5.38 1H NMR (MeOH, 300 MHz) of tryptophol (13)

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Fermentation, isolation, purification and structure elucidation

Formula: C10 H11 N O Mass: 161.200 g/mol Name: Tryptophol N H Figure 5.39 Molecular structure of Tryptophol (13)

Figure 5.40 1H NMR (MeOH, 300 MHz) of tyrosol and ferulic acid (14,15)

Formula: C10 H10 O4 Mass: 194.184 g/mol Name: Ferulic acid O Formula: C8 H10 O2 Mass: 138.163 g/mol Name: Tyrosol OH

HO HO O H C OH 3 Figure 5.41 Mol structure of Tyrosol (14) Figure 5.42 Mol structure of Ferulic acid (15)

148

Fermentation, isolation, purification and structure elucidation

15 as mixture (25.0 mg). Figure 5.37 summarizes the strategy adopted for the fermentation, isolation and purification of the active metabolites from strain SCF25.

In Fraction II compound 13 was isolated (11.8 mg) as white crystal solid with medium polarity after being subjected to PTLC 5% MeOH and column chromatography on Sephadex LH-20 (MeOH). The TLC of compound 13 showed a pink colour with anisaldehyde/sulphuric acid on heating which turns violet after two hours. MS and 1H NMR (Figure 5.38) followed by AntiBase search resulted in tryptophol (13) (Figure 5.39) which was further confirmed after comparison with literature data. As violet colour reaction with anisaldehyde/sulphuric acid on heating , as mixture compounds 14 and 15 were isolated as yellow solid (25.4 mg) after being subjected to PTLC

(CH2Cl2/5%MeOH) and column chromatography on Sephadex LH-20 1 (CH2Cl2/40%MeOH). H NMR (Figure 5.40), MS and searching in Antibase resulted in Tyrosol (14) Ferulic acid (15) (Figure 5.41-42) that were further confirmed after comparison with literature data.

Discussion

The selected Streptomyces strains were subjected to fermentation, isolation, purification and structure elucidation of the compounds. The strain RSF18 isolated from the soil of rose field yeilded three active compounds from fifteen litre broth culture. The chalcomycine a macerolide compound was isolated from the liquid phase of the broth while two cyclic thiopeptides the geninthiocine and its new derivative named as val- geninthiocine was isolated from the mycelial cake. The structure of these compounds was elucidated by mass spectrometry and NMR spectroscopy along with search in database. Yun et al., (1994) reported first time the presence of geninthiocine in the culture broth of Streptomyces sp. DD84, the compound is famous for its tip A promoter inducing activity in Stretomyces. Geninthiocin was found 16 and 64 times as active as promothiocin A and thioxamycin respectively for the induction of tipA promoter (Yun et al., 1994). The yeild of geninthiocin from Streptomyces sp. RSF18 is high, as 78.5 mg of the compound was isolated from 15 litre culture broth as compared to the already reported 71 mg from 30 litre culture broth of Streptomyces sp. DD84 by Yun et al. (1994). Thiopeptides have

149

Fermentation, isolation, purification and structure elucidation been demonstrated as antibacterial agents against Gram-positive bacteria and anaerobes, including pathogens resistant to antibiotics currently in use (Nagai et al., 2003 ; Suzumura et al., 2003), and also have potential as growth inhibitors of the human malarial parasite (Rogers et al., 1998). They were discovered as antibiotics from diverse bacteria including Streptomyces, Bacillus, and Micrococcus (Mine et al., 1972; Muir et al., 1980a). Thiopeptides have been proved later as effective growth promoters for domestic animals (Muir et al., 1980b). Most of the thiopeptide antibiotics inhibit protein synthesis in bacteria, and share a common mode of action. Thiostrepton, whose antibiotic activity is best understood, acts by binding tightly to the prokaryotic ribosome and thus inhibits translation (Ali et al., 2002). All the three purified compounds from this strain exhibited growth inhibiting activity against Gram positive bacteria and minor antifungal activity (Table 5.5). The strain CRF17 isolated from the soil of corn field was grown in twenty litre fermenter with ten litre working volume yeilded one active compound, the Alborexin. This compound was visible on TLC in prescreening as a big dark spot and in bioautography of this strain, it was the only fraction which exhibited the activity, later in preparative screening it was purified and identified as Alborexin by mass spectrometry, NMR spectroscopy and by comparison with reference data in AntiBase (Laatsch AntiBase, 2007). Alborixin is an ionophorous antibiotic of the nigericin group, it is active against Gram-positive bacteria and is coccidiostatic, but it is very toxic (Delhomme et al., 1976).The strain BG5 isolated from the soil of botanical garden yeilded almost five different compounds from twenty five litre broth culture, among them three were identified including Ochromycenone, Emycin D and 1-acetyl β-carbolin by mass spectrometry and NMR spectroscopy. Ochromycinone was first isolated by Bowie and Johnson (1967) from the fermentation broth of Streptomyces strains, it was found to exhibit enzyme inhibitory, antibacterial and antitumor activities in vitro (Guingant and Barreto, 1987). Emycin D is structurally related to ochromycinone and exhibit activity against gram-positive bacteria (Walker et al., 1999). 1-acetyl β-carbolin have been isolated from the bark of Ailanthus malabarica (Simaroubaceae), as well as from the sponge Tedania ignis (Dillman and Gardellina, 1991). The observed antifungal activity of the Streptomyces sp. BG5 in prescreening (Table 4.1, Figure 4.2) could be attributed to

150

Fermentation, isolation, purification and structure elucidation the existence of some β-carboline analogues in low concentrations, which could not be isolated. The strain CTF9 was cultivated in fifty litre fermenter (Biostat U), isolation and purification resulted in many pure fractions however most of the fractions were found to be unstable and only two fraction were identified including phenyl acetic acid and β- indole lactic acid. Phenyl acetic acid is widespread in higher plants, fungi, e.g. Aspergillus niger (Nair and Burke, 1988), and bacteria. In the microbial antibiotic production, phenyl acetic acid is added to the culture of Penecillium sp. to increase the production of penicillin G (Kato, 1953).

Strain CTF15 was cultivated as twenty five litre shaking culture, the extraction and purification of this culture broth yeilded trivial compounds including Resistomycin, Actinomycin D and Tetracenomycin D. Resistomycin was reported for the first time from the cultures of Streptomyces resistomycificus by Brockmann and Kastner (1951). Resistomycin belongs to acetogenins and is built by acetyl and malonyl units. Lee et al. (1993), reported that resistomycin had an antitumoral effect on proliferation and morphology of human breast tumor cell line MF-7. Furthermore resistomycin is active against Gram-positive bacteria preferentially inhibited the RNA synthesis in Bacillus subtilus. It is characterized by its RNA polymerase inhibitory and anti bacterial activity against Gram-positive microorganisms and Mycobacteria (Haupt et al., 1975). The orange-red actinomycins are a family of chromo peptide anti tumor antibiotics, isolated from different Streptomyces strains, of which 70 native and many synthetic variants are known (Brockmann, 1960; Waksman, 1968). They are known since 1940 when Waksman and Woodruff (1940), isolated actinomycin A, as first ever crystalline antibiotic. Actinomycin complexes termed as A, B, C, D, I, X, Z as well as other actinomycin analogues have been reviewed (Lackner et al., 2000). The natural actinomycins all share the same phenoxazinone chromophore, varying only in the amino acid content of their two depsipentapeptide moieties. Actinomycins C3 and D have found clinical applications as anticancer drugs, particularly in the therapy of Wilm’s tumor (Green, 1997) and soft tissue sarcoma (Womer, 1997) of children, and are still of interest in molecular biology (Wadkins et al., 1998). Recently actinomycin D has been proposed as therapeutic agent for AIDS, because it is a potent inhibitor of HIV-1 minus strand transfer (Guo et al., 1998).

151

Fermentation, isolation, purification and structure elucidation

The strain SCF25 was grown in fifty litre fermenter, extraction and purification of this broth culture yeilded many fractions which were difficult to purify and were unstable, however three fractions were identified from this strain including Tryptophol, Ferulic acid and Tyrosol. Tryptophol and Tyrosol were reported to be antibiotically weakly active against Saccharomyces cerevisiae, Nematospora corlyi, and are moderately phytotoxic and antifungal (Laatsch, 2003). Tyrosol is widespread in fungi e.g. Candida sp. (Lingappa et al., 1969), Ceratocystis fimbriata, Cochliobolus lunata, G. fuikuroi, Pyricularia oryzae as well as several Ceratocystis sp. isolated from leaves of Ligustrum ovalifolium and the bark of Fraxinus excelsior, or Gibberella fuikuroi and peanuts (Cross et al., 1963).

It is worth mentioning here that the strains cultivated as shaking cultures in flasks on a linear shaker gave better results as compared to the strains cultivated in a fermenter. The strains RSF18, BG5 and CTF15 cultivated as shaking cultures yeilded higher quantities of crude extracts and working up of these crude extracts yeilded highly pure and stable fractions on which the structure elucidations were performed. While the strains CRF14, CTF9 and SCF25 produced small quantities of the crude extract which were difficult to purify and identify because most of the pure fractions obtained were found to be unstable. It may be attributed to the difference in general culture conditions in a shaking flask and a stirring fermenter.

152

16s rRNA gene sequencing

CHAPTER 6 16S rRNA GENE SEQUENCE ANALYSIS The sequence of the 16S rRNA gene has been widely used as a molecular clock to estimate relationships among bacteria (phylogeny), but more recently it has also become important as a means to identify an unknown bacterium up to the genus or species level (Sacchi et al., 2002). Advances have been made in automating and minimizing the detection times using biochemical methods, however, biochemical identification is not accurate for determining the genotypic differences of microorganisms. A more accurate method for genotype determination is that of the molecular biological approach of ribotyping by comparing similarities in the rRNA gene sequences. The sequence data for the 16S rRNA gene is highly conserved for different organisms and has also been shown to be very accurate for genus and species identification of eubacteria. Carl Woese and colleagues used first of all the rRNA sequence data to examine evolutionary relationships among bacteria by comparing ribonuclease T1-generated oligonucleotides (Woese et al., 1990). Subsequently, a number of authors have identified/constructed and published phylogenetic trees based on 16S rRNA gene sequences (Sass and Cypionka, 2004). Comparisons of the sequences between different species suggest the degree to which they are related to each other, a relatively greater or lesser difference between two species suggests a relatively earlier or later time in which they shared a common ancestor. The 16S rRNA has properties, which predestine it as a universal phylogenetic marker. There are regions on the 16S rRNA that are quite conserved and others, which are variable. Comparing the differences in the base sequence of this 16S rRNA gene is, therefore, an excellent means to study evolutionary changes and phylogenetic relatedness of organisms. Hence genotypic classification based on nucleotide sequence comparison of 16S rRNA genes has become available as additional taxonomic tool. Using this new standard, phylogenetic trees, based on base differences between species, are constructed, and bacteria are classified and reclassified into new genera. The advantage of 16S rRNA gene analysis is also that it can potentially be applied to the identification of all bacteria. Molecular identification techniques provide two primary advantages to phenotypic identification, a more rapid turnaround time and improved accuracy in identification (Springer et al., 1996; Gupta, 2004). Molecular phylogeny increasingly supports the

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16s rRNA gene sequencing understanding of organismic relationships and provides the basis for the classification of microorganisms according to their natural affiliations. Identification of Streptomyces has traditionally been accomplished by several techniques, including selective plating methods (Kuester and Williams, 1964), the proof of the presence of L, L-diaminopimelic acid, the absence of characteristic sugars in the cell wall (Lechevalier and Lechevalier, 1970) and the construction of genetic marker systems (Wipat et al., 1991). The 16S rRNA gene sequence data have proved invaluable in sreptomycetes systematic, in which they have been used to identify several newly isolated species (Mehling et al., 1995; Pace, 1997; Nelson et al., 2001).

In this study the final taxonomic status of the selected Streptomyces strains was determined by ribotyping. From the twenty strains selected for detailed studies seventeen strains were selected depending upon the prescreening and preparative screening results and were identified up to species level. Among the corn field isolates, the strains CRF11, CRF14, CRF17 exhibited almost similar type of biological activities (Table 4.1) and same metabolic profile (Table 4.2; Fig 4.3) in chemical screening, so only the isolates CRF11 and CRF17 were selected for ribotyping. Similarly among the cotton field isolates, the strains CTF23 and CTF24 exhibited minor biological activities (Table 4.1) and their metabolic profiles seemed uninteresting in chemical screening (Table 4.2; Fig 4.3), so these strains were ignored in ribotyping studies. The genomic DNA of the selected strains was isolated following the methods of Felnagle et al. 2007. The primers used for the amplification of 16S rRNA gene, the reaction mixture and the PCR conditions are mentioned in Tables 2.50-2.52. The PCR product was purified by gel extraction and was sequenced (the detailed procedure adopted for DNA extraction and sequencing is given in chapter 2). The sequences obtained by the forward primer P1 (Table 2.50) and the reverse primer P2 (Table 2.50) were refined and the readable regions of both sequences were joined together (by taking the reverse complement of the second sequence) to get the maximum sequence of the 16S rRNA gene. The sequence of the 16S rRNA gene of each isolate was analyzed using the advanced BLAST search program at the NCBI website: http://www.ncbi.nlm.nih.gov/BLAST/. The BLAST analysis of 16S rRNA gene sequences of the selected strains showed alignments of these sequences with reported 16S rRNA genes in the gene bank. The number of nucleotides sequenced for

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Table 6.1: Gene bank accession numbers along with the alignments of sequences obtained with reported 16S rRNA gene sequences in the gene bank and highest similarity with different Streptomyces species (17 strains).

No. of Nucleotides Gene bank Isolate % similarity with sequenced (bp) Accession Number

RSF 17 1421 Streptomyces sp. 99 EU301834

RSF 18 1418 Streptomyces sp. 99 EU294139

RSF 23 976 S. chromofuscus 94 EU301837

BG 5 1433 S. matensis 98 EU301836

CRF 1 721 S. macrosporeus 99 EU301829

CRF 2 1437 S. vinaceus 99 EU294133

CRF 11 859 S. heliomycini 97 EU301828

CRF 17 1420 S. pulcher 98 EU294134

SCF 18 1417 S. griseoincarnatus 98 EU294140

SCF 25 1425 S. macrosporeus 99 EU301835

SCF 31 867 S. malachitofuscus 85 EU294141

CTF 9 1426 S. malachitofuscus 99 EU294138

CTF 13 1390 S. erythrogriseus 99 EU301830

CTF 14 1393 S.labedae 99 EU294135

CTF 15 1429 S.griseoincarnatus 99 EU301831

CTF 20 1427 S. rochei 99 EU294136

CTF 25 1425 S. macrosporeus 98 EU301833

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16s rRNA gene sequencing each strain and the highest similarities found with different species of the genus Streptomyces are summarized in Table 6.1. The nucleotide sequence data was deposited to the gene bank and gene bank accession number for each strain was obtained (Table 6.1). The 16S rRNA nucleotide sequences of the isolates were aligned with homologous regions from various actinomycetes, and the phylogenetic trees were constructed by neighbor-joining method (Saitou and Nei 1987). A bootstrap confidence analysis was performed on 1000 replicates to determine the reliability of the distance tree topologies obtained (Felsenstein, 1985). The graphic representation of the resulting tree was obtained by using DAMBE (Data Analysis in Molecular Biology and Evolution) software (Version 4.0.36). The sample and the top 2-5 matches in the gene bank are included in the phylogenetic tree of each strain, which visually illustrates the relatedness of the organisms, and allows one to estimate the average genetic distance within the closely related matches (Fig 6.3-6.19). As an example, the 16S rRNA gene sequence of the one representative strain (isolate RSF17) consisting of 1409 bp, as plane text is given in Fig 6.1. The electropharogram of the sequences of the isolate RSF17 obtained by forward and reverse primers are given in Fig 6.2. The 16S rRNA gene sequences of the remaining isolates in the form of electropharogram are given as the suplimental data at the end of the thesis.

On the basis of Phylogenetic data obtained the strains RSF17 and RSF18 showed maximum similarity (99%) with Streptomyces sp. H-9 and Streptomyces sp. 3-97 respectively, however the homology of 97% of both of these strains was obtained with Streptomyces variabilis strain 173733 in BLAST analysis. The Phylogenetic trees of Strains RSF17 and RSF18 constructed by neighbor joining method using 1000 bootstrap replicates show their genetic relationships with top 4-5 matches in the gene bank (Fig: 6.3-6.4). The strain RSF23 showed maximum homology (94%) with Streptomyces chromofuscus strain NRRL B-12175, however the homology of 93% was found with Streptomyces nogalator and Streptomyces achromogens. The phylogenetic tree of the strain RSF23 showing genetic relationship with top four matches in the gene bank is shown in Fig 6.5. The strain BG5 showed maximum similarity (98%) with Streptomyces matansis, the other closest matches included,

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Streptomyces tandae strain 173569 (97%) and Streptomyces griseoflavus strain 174490 (97%), its Phylogenetic tree was constructed by neighbor joining method using 1000 bootstrap replicates confirms its genetic similarity with Streptomyces matansis (Fig 6.6). Among the corn field isolates, the strains CRF1 exhibited maximum similarity (99%) with Streptomyces macrosporeus strain 52, Streptomyces macrosporeus strain Q192 (99%) and Streptomyces macrosporeus strain 14312 (99%) (Fig 6.7). The strain CRF2 exhibited maximum similarity (99%) with Streptomyces vinaceus, its phylogenetic tree showing its relation to the closest matches is given in Fig 6.8. Similarly the strains CRF11 and CRF17 exhibited maximum similarity with, Streptomyces heliomycini (97%), and Streptomyces pulcher (98%) respectively, in BLAST analysis. Their Phylogenetic tree constructed by neighbor joining method confirms their genetic relatedness with these strains (Fig 6.9, 6.10). The strains SCF18, SCF25, SCF31 isolated from the sugar cane fields exhibited maximum similarity with Streptomyces griseoincarnatus (98%), Streptomyces macrosporius (99%) and Streptomyces malachitofuscus (85%) respectively, in BLAST analysis. Their Phylogenetic trees showing their genetic relatedness with 2-3 top matches in the gene bank are given in the Figures 6.11, 6.12, and 6.13. Among the cotton field isolates, the strain CTF9 exhibited maximum similarity (99%) with Streptomyces malachitofuscus, the other closely organism is Streptomyces sp. 210741 (97%), its phylogenetic tree is shown in Figure 6.14. The strains CTF13 and CTF14 exhibited close resemblance with Streptomyces erythrogriseus (99%) and Streptomyces labedae (99%) respectively, their phylogenetic trees exhibiting their resemblance with the top 4-5 matches in gene bank are given in Figures 6.15 and 6.16. Similarly the isolate CTF15 exhibited maximum similarity (99%) with Streptomyces griseoincarnatus, the other closely related organisms includes Streptomyces sp. CHR3 and Streptomyces sp. A446Ydz-DS (Fig 6.17). The other strains of this group CTF20, and CTF25 exhibited maximum similarity with Streptomyces rochei (99%), and Streptomyces macrosporeus (98%) respectively, in BLAST analysis. Their Phylogenetic trees constructed by Neighbor Joining method showing their relation with top 4-5 matches in the gene bank are given in Fig 6.18-6.19.

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Fig: 6.1 16S rRNA gene sequence of the Streptomyces sp. RSF17

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B Figure 6.2 Electropharogram of the 16S rRNA gene sequence of the isolate RSF17, A= sequence obtained by the forward primer P1, B= sequence obtained by the reverse primer P2

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N. Joining Tree

EF 199764 Streptomyces sp. 3-97 EU 570717 Streptomyces variabilis strain 173733 EU 570433 Streptomyces variabilis strain 173491 EF 675608 Streptomyces sp. H-9 EU 301834 Streptomyces sp. strain RSF17

Fig: 6.3 Phylogenetic Tree of Streptomyces sp. Strain RSF17

N. Joining Tree

EF675608 Streptomyces sp. H7 EU570717 Streptomyces variabilis strain 173733 EU570433 Streptomyces variabilis strain 173491 EF199764 Streptomyces sp. 3-97 EU294139 Streptomyces sp. strain RSF18

Fig: 6.4 Phylogenetic Tree of Streptomyces sp. Strain RSF18

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N. Joining Tree

AB184194 Streptomyces chromofuscus AY999800 Streptomyces chromofuscus strain NRRL B-12175 EU301837 Streptomyces chromofuscus strain RSF23 AB184408 Streptomyces nogalater AB184562 Streptomyces achromogenes subsp. Streptozoticus

Fig: 6.5 Phylogenetic Tree of Streptomyces chromofuscus Strain RSF23

N. Joining Tree

EU301836 Streptomyces matensis strain BG5 AB184221 Streptomyces matensis EU570499 Streptomyces tendae strain 173569 EU593701 Streptomyces griseoflavus strain 174490

Fig: 6.6 Phylogenetic Tree of Streptomyces matensis Strain BG5

N. Joining Tree

EU301829 Streptomyces macrosporeus strain CRF1 EF063469 Streptomyces macrosporeus strain 52 EF063494 Streptomyces macrosporeus strain Q192 EF371436 Streptomyces macrosporeus strain 14312

Fig: 6.7 Phylogenetic Tree of Streptomyces macrosporeus Strain CRF1

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N. Joining Tree

EU294133 Streptomyces vinaceus strain CRF2 AB184763 Streptomyces vinaceus AB184186 Streptomyces vinaceus

Fig: 6.8 Phylogenetic Tree of Strain Streptomyces vinaceus Strain CRF2

N. Joining Tree

EU301828 Streptomyces heliomycini strain CRF11 EU593729 Streptomyces heliomycini strain 173574 EU570432 Streptomyces heliomycini strain 173689 EU570416 Streptomyces heliomycini strain 173895

Fig: 6.9 Phylogenetic Tree of Streptomyces heliomycini Strain CRF11

N. Joining Tree

EU294134 Streptomyces pulcher strain CRF17 EF063494 Streptomyces macrosporeus AF452714 Streptomyces speibonae AB184422 Streptomyces pulcher

Fig: 6.10 Phylogenetic Tree of Streptomyces pulcher Strain CRF17

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N. Joining Tree

EU294140 Streptomyces griseoincarnatus strain SCF18 AB184884 Streptomyces variabilis EU570412 Streptomyces variabilis strain 173893 EU301831 Streptomyces griseoincarnatus strain CTF15

Fig: 6.11 Phylogenetic Tree of Streptomyces griseoincarnatus Strain SCF18

N. Joining Tree

EU301835 Streptomyces macrosporeus strain SCF25 EF063469 Streptomyces macrosporeus strain 52 EF063494 Streptomyces macrosporeus strain Q192 AB184542 Streptomyces macrosporeus

Fig: 6.12 Phylogenetic Tree of Streptomyces macrosporeus Strain SCF25

N. Joining Tree

EU294141 Streptomyces malachitofuscus strain SCF31 AB184282 Streptomyces malachitofuscus AJ781347 Streptomyces malachitofuscus EF424396 Streptomyces sp. SL-2

Fig: 6.13 Phylogenetic Tree of Streptomyces malachitofuscus Strain SCF31

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N. Joining Tree

EU196533 Streptomyces malachitofuscus clone B16 AJ781347 Streptomyces malachitofuscus AB184282 Streptomyces malachitofuscus EU600088 Streptomyces sp. 210741 EU294138 Streptomyces malachitofuscus strain CTF9

Fig: 6.14 Phylogenetic Tree of Streptomyces malachitofuscus Strain CTF9

N. Joining Tree

EU301830 Streptomyces erythrogriseus strain CTF13 AB184605 Streptomyces erythrogriseus DQ663180 Streptomyces sp. 3001 AJ781328 Streptomyces erythrogriseus

Fig: 6.15 Phylogenetic Tree of Streptomyces erythrogriseus Strain CTF13

N. Joining Tree

EU294135 Streptomyces labedae strain CTF14 AY999868 Streptomyces labedae AJ781321 Streptomyces griseoincarnatus AB184704 Streptomyces labedae Fig: 6.16 Phylogenetic Tree of Streptomyces labedae Strain CTF14

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N. Joining Tree

EU301831 Streptomyces griseoincarnatus strain CTF15 AB184207 Streptomyces griseoincarnatus AJ781321 Streptomyces griseoincarnatus AF026080 Streptomyces sp. CHR3 EU257265 Streptomyces sp. A446Ydz-DS

Fig: 6.17 Phylogenetic Tree of Streptomyces griseoincarnatus strain CTF15

N. Joining Tree

EU294136 Streptomyces rochei strain CTF20 EU593731 Streptomyces rochei strain 173672 DQ026641 Streptomyces enissocaesilis strain NRRL B-16365 DQ663150 Streptomyces sp. 3194 EF626598 Streptomyces rochei strain NRRL B-1559

Fig: 6.18 Phylogenetic Tree of Streptomyces rochei Strain CTF20

N. Joining Tree

EU301833 Streptomyces macrosporeus strain CTF25 EF063494 Streptomyces macrosporeus strain Q192 EF063469 Streptomyces macrosporeus strain 52 DQ663148 Streptomyces sp. 3187 AB184542 Streptomyces macrosporeus

Fig: 6.19 Phylogenetic Tree of Streptomyces macrosporeus strain CTF25

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DISCUSSION A whole array of taxonomic tools has been used to define genera and suprageneric groups of actinomycetes, but partial sequence analysis of 16S ribosomal RNA is the most significant (Goodfellow, 1989; Wellington et al., 1992). Traditional methods of actinomycetes taxonomy relied heavily upon morphological, biochemical and physiological characteristics. Currently, there is an increased interest in applying molecular genetics techniques to resolve many of the issues and problems created by the present state of actinomycetes taxonomy. Within the last decade, a number of workers began using molecular techniques to answer questions dealing with the taxonomy, population dynamics, and the evolution of this group of bacteria. Among the most popular molecular techniques employed are DNA-DNA hybridization, sequence determination of small ribosomal subunit ribonucleic acids (16S rRNAs), and to a lesser degree, restriction fragment length polymorphism (RFLP) analysis of DNA (Williams et al., 1989; Wellington et al., 1992). Molecular techniques involving ribosomal RNA sequence analysis are commonly used to investigate evolutionary relationships within different genera of bacteria. Although rRNA sequence data does not provide the taxonomic resolving power seen with RFLPs and DNA-DNA hybridization, it is nonetheless advantageous in that it is a single-step experiment (Turner, 1997). In general, information obtained using molecular techniques is very useful in that it provides researchers with a powerful and independent data set in which hypotheses generated from other data, such as morphology and physiology, can be tested (Li et al., 2000). In a routine antibiotic screening strategy, the strains showing interesting activity or producing interesting metabolites in preparative screening are usually identified up to species level. Seventeen Streptomyces strains which exhibited interesting results in prescreening and preparative screening were identified by 16S rRNA gene sequencing. The morphological, biochemical and physiological characterization of these strains strongly suggested that they are the members of genus Streptomyces. In ribotyping studies these strains exhibited genetic similarity with different species of the genus Streptomyces and in most of the cases similarity up to 99% was found. It has been demonstrated that 16S rRNA gene sequence data of an individual strain with a nearest neighbor exhibiting a similarity score of <97% represents a new species, the meaning of

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16s rRNA gene sequencing similarity scores of >97% is not as clear (Petti, 2007). In most of the cases in this ribotyping study, the 16s rRNA gene sequence up to 1400 bp was obtained (Table 6.1), which increases the certainty of the resemblance of an isolate with a particular Streptomyces specie. The isolates RSF23 and CRF11 exhibited 94% and 97% homology with Streptomyces chromofuscus and Streptomyces heliomycini respectively (Table 6.1), which indicate that they may be the same species or may be the different ones. Similarly another isolate, the strain SCF31 exhibited 85% homology with Streptomyces malachitofuscus, which also indicate that it may also be a separate specie, however in these three isolates the number of nucleotides sequenced was low i.e. less than 1000 bp (Table 6.1), this might be the reason for their low genetic similarity with the reported Streptomyces species and the complete 16s rRNA gene sequence of these isolates may clear their taxonomic position. The remaining all the isolates including the six strain RSF18, BG5, CRF17, SCF25, CTF9, CTF15, which were used in fermentation studies and the metabolites produced by them were identified in preparative screening, exhibited more than 98 % homology with different species of the genus Streptomyces (Table 6.1), so they can be considered as the same species, because the comparison of their morphologic, biochemical and physiological characteristics with those of known Streptomyces species described in Bergey’s Manual of Systematic Bacteriology (Lechevalier et al., 1989), also exhibited close similarity of these isolates with the same species up to some extent. The sequences of 16S rRNA genes of all the identified strains are available in gene bank with the Accession Numbers mentioned in Table 6.1. The Phylogenetic trees of all the seventeen strains based on 16s rRNA gene sequences were constructed by neighbor joining method using 1000 bootstrap replicates, which show the genetic relatedness of each of these strains with top 4-5 matches in the gene bank (Fig 6.3-6.19). An example electropharogram of the 16s rRNA gene sequence of the isolate RSF17 has been included in the results, while the electropharograms of all the remaining isolates are given in the supplemental data.

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CHAPTER 7

CULTURE OPTIMIZATION

Media used in cultivation of microorganisms must contain all elements in a form suitable for the synthesis of cell substances and for the production of metabolic products. An optimally balanced culture medium is mandatory for maximal production of a target compound. Different strategies can be used for optimizing cultivation conditions of bacteria however the conventional “one variable at a time” approach is used more often than other approaches (Mao et al., 2007; Farid et al., 2000). Some statistical techniques, such as Plackett-Burman design and response surface methodology (RSM), were extensively used in the industries and in bioprocesses including the formulation of culture medium for bacteria and fungi (Didier et al., 2007). The isolates RSF 18 and CTF 9 were selected in order to find out the appropriate medium and optimal culture conditions for the growth and maximum production of the metabolites produced by them. Six different media compositions as given in Table 2.31- 2.37, were used in order to find out the best medium composition for the growth of these Streptomyces strains. Along with the different media compositions, following four sets of different culture conditions were applied to determine the appropriate culture conditions. CC1: pH 6.5, incubation temperature 28oC, shaking at 95 rpm, for 7 days. CC2: pH 6.5, incubation temperature 35oC, shaking at 110 rpm, for 7 days. CC3: pH 7.8, incubation temperature 28oC, shaking at 95 rpm, for 7 days. CC4: pH 7.8, incubation temperature 35oC, shaking at 110 rpm, for 7 days.

Three litres of each of the six media were dispensed into twelve one litre flasks (250 ml broth in each flask). The flasks were arranged into groups according to culture conditions, so that under each culture condition there were three replicates R1, R2 and R3 (three one litre flasks containing 250 ml media each), the pH was adjusted in each flask separately. After autoclaving, the flasks were inoculated by test strains from well grown plates and were incubated at the proposed culture conditions CC1, CC2, CC3 and CC4. The strains were harvested after seven days and the culture broth in each of the flask was freeze dried and was extracted individually three times with ethyl acetate. The crude

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Culture Optimization extracts were obtained after evaporating the ethyl acetate on a rotary evaporator. Two parameters i.e. weight of the crude extract/ 250 ml of the culture broth and antimicrobial activity of the extracts against a set of test organisms (Bacillus subtilus, Staphylococcus aureus and Streptomyces viridochromogens TU57) were considered for the evaluation of the results. Mean weight of the crude extracts was determined for strains RSF18 and CTF9 grown on six different media under variable conditions of pH, temperature and aeration. The results obtained were analysed statistically applying analysis of variance (ANOVA) test (Duncan’s multiple range test) using SPSS software version 10 at (P=0.001).

Impact of different culture conditions on the growth of strain RSF18 in a particular culture medium

In medium A, the culture condition CC1 has significant impact on the growth of strain RSF18 and yielded maximum crude extract as compared to all other culture conditions employed. The culture condition CC3 manifested a less significant impact as compared to CC1 but induced a significant increment in the crude weight profile of RSF18 as compared to CC2 and CC4, which both have presented a similar impact on the growth (Table 7.1). In medium B, the culture condition CC3 significantly enhanced the growth by increasing the crude extract yield of the strain when compared to the crude extract yield under culture conditions CC4, CC1 and CC2. A comparably profound increment in the crude extract weight was observed under the culture condition CC4 than CC1 and CC2 conditions (Table 7.1). When compared to each other the conditions CC3 and CC4 in the medium C had induced more or less similar insignificant effect on the growth of the strain RSF18 but the increase in the weight of crude extract is significantly high in comparison to the yield under CC1 and CC2 conditions. Significant increment in the weight yield had been produced by the culture condition CC1 as compared to the CC2 condition (Table 7.1). In medium D, the growth significantly differed under all the four culture conditions, being highest at the culture condition CC4 (Table 7.1). In medium E, there was an insignificant difference between the yields under the culture conditions CC2 and CC3. The estimated crude weight yields under both of these conditions were however significantly high as compared to those under culture conditions CC4 and CC1

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(Table 7.1). In medium F, all the four culture conditions brought about a significant difference in the crude weight profile of the strain .The culture condition CC3 yielded maximum quantity of the crude extract as compared to the yields at culture conditions CC4, CC2 and CC1. The setup CC3 is the second most favourable culture condition inducing an increase in the crude extract weight of the strain as compared to the culture conditions CC1 and CC2 (Table 7.1).

Impact of different culture media on the growth of strain RSF18 under a particular set of culture conditions

In case of culture conditions CC1, the strain RSF18 exhibited most prominent growth on medium D and yielded maximum quantity of the crude extract as compared to all other media employed. The media A and B had similar impact on the growth with no significant difference among them, however they showed high significant difference as compared to the media F, E and C (Table 7.1). Under culture conditions CC2, the medium D had a significant impact on the growth as compared to all other media. The media E and F had more or less similar impact on the growth with an insignificant difference as compared to each other. The growth of the respective strain on media E and F was highly significant when compared with the growth in the media A, B and C. Medium C was the medium with the least significant impact on the growth of the strain RSF18 and yielded minimum quantity of the crude extract under culture conditions CC2 (Table 7.1). In culture conditions CC3, all the media A, B, C, D, E and F had significantly different impact on the growth of the strain RSF18 with medium D possessing the maximum impact as compared to the rest of three media and the medium C having the minimum impact on the growth (Table 7.1). Considering the case of culture conditions CC4, the maximum amount of crude extract was obtained from medium D as compared to all other media employed, the medium B provided the second highest yield as compared to the media F, E, A and C (Table 7.1). Eventually under all the four sets of culture conditions (CC1, CC2, CC3 and CC4) medium D yielded maximum quantity of the crude extract and the medium C yielded minimum quantity of the crude extract of strain RSF18 (Table 7.1).

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Table 7.1: Comparison of the Wt. of crude extracts/250 ml culture broth of the strains RSF 18 and CTF9 for six different media compositions and four different sets of culture conditions (Mean of 3 replicates taken ± S.E)

Strains Culture conditions Types of Media Med A Med B Med C Med D Med E Med F CC1 37.17bw± 0.725 38.28by±0.765 5.42ex±0.159 154.14ax±0.834 9.30dz±0.173 17.52cz±0.159 RSF18 CC2 5.05dy±.0.144 22.64cz±0.150 2.97ey±0.176 144.48ay±0.499 28.51bw±0.514 28.52by±0.300 CC3 23.12ex± 0.254 67.28bw±0.906 9.37fw±0.159 130.38az±1.031 27.92dw±0.528 62.94cw±0.644 CC4 5.26fy± 0.147 61.82bx±0.473 8.31ew±0.283 181.15aw±0.664 20.75dx±0.375 54.42cx±0.243 Med A Med B Med C Med D Med E Med F CC1 22.65ey±0.805 26.61dy±0.352 25.75dx±1.014 96.66cz±.0.476 166.68ax±0.644 151.50by±0.231 CTF9 CC2 32.593fx±1.273 51.61cx±1.951 34.41ew±0.231 213.06aw±0.669 37.32dy±1.544 161.05bx±1.187 CC3 13.793ez±0.453 26.62dy±0.473 26.61dx±0.929 106.36cy±1.509 171.63bw±0.918 183.50aw±1.097 CC4 64.27cw±0.274 58.90dw±1.674 36.55fw±0.199 150.87ax±0.502 40.08ey±1.319 127.29bz±1.207

Values designated by a, b, c, d, e, f are for six different media compositions, Values designated by w, x, y, z are for four different culture conditions, Values designated with the same alphabets are not significantly different from each other, Duncan’s (1955) multiple range test at Significance level = 0.001

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Impact of different culture conditions on the growth of strain CTF9 in a particular culture medium

In media A and B, under the culture conditions CC4 maximum significant effect on the growth of the strain CTF9 was noticed as compared to the other culture conditions i.e CC1,CC2 and CC3. The culture condition CC2 had a relatively more profound impact on the yield of the strain as compared to the culture conditions CC1 and CC3 (Table 7.1). In media B and C the culture conditions CC2 has significant impact on the growth and yielded maximum crude extracts as compared to the other culture conditions. A significant increment with maximum growth yield was obtained in media C and D when the strain was cultured under the condition CC4 and CC2 respectively. Relative significance of the rest of the three culture conditions according to effect on the yield of the strain CTF9 is CC2>CC3>CC1 for the medium C and CC4>CC3>CC1 for the medium D (Table 7.1). In media E and F, the culture conditions CC3 has significant impact and yielded maximum quantity of the crude extracts as compared to the all other culture conditions (CC1, CC2 and CC4) employed for the growth of the strain CTF 9 (Table 7.1).

Impact of different media on the growth of strain CTF9 under a particular set of culture conditions

The media E and F, had significant impact on the growth with the maximum increase in the yield of strain CTF9 under culture conditions CC1 as compared to other culture media A, B, C and D (Table 7.1). Under culture condition CC2, the medium D had significant impact on the growth followed by the medium F which induced the second maximum yield after the medium D (Table 7.1). Under culture conditions CC3, medium F had significant impact with maximum enhancement in the crude weight yield of the strain as compared to others. Following medium F, a significant impact on the growth was also noticed in media E and D. (Table 7.1). Under culture conditions CC4, the medium D had significant impact on the growth, after this medium F caused a significant increase in the growth of strain CTF9 as compared to the media A, B, C and E (Table 7.1).

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Impact of different culture conditions on the antimicrobial activity of the strain RSF18 in a particular culture medium

The crude extracts obtained from different culture media (A, B, C, D, E, F) under four different sets of culture conditions (CC1, CC2, CC3 and CC4) were analysed for their antimicrobial activity against three test organisms (Bacillus subtilus, Staphyloccocus aureus, Streptomyces viridochromogens Tü 57). In medium A, the strain RSF18 exhibited the maximum antimicrobial activity against the test organisms B. subtilus, S. aureus, S. viridochromogens Tü 57, under the culture conditions CC1 and CC3 (Table 7.2). In medium B, under the culture condition CC1 high profile inhibitory mode of action against test organisms was observed (Table 7.2). In case of medium C, a maximum significant antimicrobial effect was noticed under the culture condition CC2 against the strains B. subtilus, S. aureus, whereas for the test organism S. viridochromogens Tü 57, the maximum antimicrobial activity was observed under the culture condition CC1 (Table 7.2). In medium D, the antimicrobial activity of the strain RSF18 was more profound under the culture conditions CC3 as compared to all others conditions, for all the test organisms with the exception of S. viridochromogens Tü 57, against which the maximum inhibitory activity was observed under the culture condition (Table 7.2). In case of medium E, the culture conditions CC2 and CC4 provided a significant antimicrobial activity attribute to the strain RSF18 against the test organisms (Table 7.2). Similarly in case of medium F, the culture condition CC2 had a significant impact on the antimicrobial activity (Table 7.2).

Impact of different media on the antimicrobial activity of the strain RSF18 under a particular set of culture conditions

In the case of culture condition CC1, the strain RSF18 exhibited significantly higher antimicrobial activity in the media A and D, as compared to the antimicrobial activity profile displayed by RSF18 in the presence of other media B, C, E and F, (Table 7.2). Under culture conditions CC2, in the media F, the strain showed significantly higher antimicrobial activity, followed by the media E and C which contributed the maximum antimicrobial attribute to the strain RSF18 against the test organisms (Table 7.2). The strain RSF18, in the medium A and D and F under the culture condition CC3, acquired a significant antimicrobial activity while the other three media B, C and E failed to provoke

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Table 7.2: Comparison of the antimicrobial activity of the strains RSF18 and CTF9 at six different media compositions and four different sets of culture conditions (Mean of 3 replicates taken ± S.E) Strain RSF18 Antimicrobial activity test organisms Zone of inhibition (mm) B.subtilus S.aureus S. virido chromogenes (Tü 57) Media / Culture CC1 CC2 CC3 CC4 CC1 CC2 CC3 CC4 CC1 CC2 CC3 CC4 conditions Med A 15±0.000aw 10±0.333dy 15±0.000aw 12±0.000bx 13±0.000aw 0±0.000dy 13±0.00bw 11±0.000dx 20±0.000aw 16±0.577bx 20±0.000aw 15±0.000bz Med B 13.6±0.33bw 11±0.000cx 0±0.000dy 11±0.000cx 11.6±0.33bw 0±0.000dx 0±0.000cx 10.6±0.33dw 15.6±0.33cw 12.6±0.33dy 10.6±0.33cz 14±0.000cx Med C 12.6±0.33cx 14±0.000bw 0±0.000dy 12±0.000bx 10±0.000cx 17.6±0.33aw 0±0.000cy 16.6±0.33aw 18±0.000bw 15.6±0.333cx 11±0.000cz 13±0.333dy Med D 11±0.000dx 11±0.000cx 14±0.000bw 10.6±0.33cx 12±0.000bx 10±0.577cy 15±0.000aw 11±0.000dc 20±0.000aw 11±0.000ey 20±0.000aw 12±0.000ex Med E 11±0.000dx 14.6±0.33bw 0±0.000dy 14±0.000aw 10.6±0.333cy 17±0.000aw 0±0.000cz 14.6±0.333bx 13±0.000dx 18.6±0.00aw 0±0.000dy 18±0.000aw Med F 0±0.000ey 15.6±0.33aw 11±0.000cx 11±0.000cx 0±0.000dx 12±0.000bw 0±0.000cx 12.6±0.33cw 0±0.000ey 16.6±0.33bw 12±0.000bx 12.6±0.33dx Strain CTF9 Antimicrobial activity test organisms Zone of inhibition (mm) B.subtilus S.aureus S. virido chromogenes (Tü 57) Media / Culture CC1 CC2 CC3 CC4 CC1 CC2 CC3 CC4 CC1 CC2 CC3 CC4 conditions Med A 0±0.000cy 17.6±0.33bx 0±0.000cy 19.6±0.33aw 0±0.000dx 12±0.577cw 0±0.000dx 13±0.000aw 0±0.000cy 21.6±0.33aw 0±0.000ey 17.6±0.33bx Med B 13±0.000ax 0±0.000ez 16.6±0.33aw 11±0.000cy 10.6±0.333bx 0±0.00dy 12±0.000bw 11±0.000bx 16±0.000bx 12±0.000dy 17.6±0.33aw 12.6±0.33dy Med C 0±0.000cx 11.6±0.33cw 0±0.000cx 0±0.000dx 0±0.000dy 14.6±0.33aw 11±0.000cx 0±0.000cy 0±0.000cy 20±0.000bw 0±0.000ey 12±0.000dx Med D 11±0.000bx 10.6±0.33dx 12±0.000bw 11±0.000cx 12.6±0.33aw 13±0.000bw 11±0.000cx 13±0.000aw 18±0.000aw 17.6±0.33cw 14±0.000cy 15±0.000cx Med E 0±0.000cx 19.6±0.33aw 0±0.000cx 20±0.000aw 9.6±0.333cx 13±0.000bw 0±0.000dy 13±0.000aw 0±0.000cz 17.6±0.333cx 10±0.000dy 21±0.000aw Med F 0±0.000cy 0±0.000ey 12.6±0.33bx 16.6±0.33bw 0±0.000dy 0±0.000dy 15.6±0.33aw 11±0.000bx 0±0.000cy 0±0.000ey 16.6±0.33bw 14.6±0.33cx Values designated by a, b, c, d, e, f are for six different media compositions, Values designated by w, x, y, z are for four different culture conditions, Values designated with the same alphabets are not significantly different from each other, Duncan’s (1955) multiple range test at Significance level = 0.001

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any antimicrobial activity in the strain (Table 7.2). Under culture conditions CC4, the medium E has significant impact on the antimicrobial activity as compared to other media, the impact of media A, B, C, D and F on the antimicrobial activity was more or less similar (Table 7.2).

Impact of different culture conditions on the antimicrobial activity of the strain CTF9 in a particular culture medium

In case of medium A, the culture conditions CC2 and CC4 had significant impact on the antimicrobial activity of the strain CTF9 as compared to the antimicrobial activity obtained under the culture conditions CC1 and CC3 (Table 7.2). In medium B, the culture conditions CC3 had a significant impact on the antimicrobial activity as compared to the other culture conditions employed. Under the culture condition CC2, the strain CTF9 either showed low level of antimicrobial activity (S. viridochromogens Tü 57) as compared to the other culture conditions or refrained to impose any inhibitory effect against the test organism (B. subtilus , S. aureus) (Table 7.2). In medium C, the culture conditions CC2 had significant impact on the antimicrobial activity as compared to the other culture conditions (Table 7.2). In the medium D, the culture conditions CC1 and CC2 had a significant impact on the antimicrobial activity of the strain CTF9 as compared to the culture conditions CC3 and CC4. In medium E, under the culture condition CC4, the strain exhibited significantly high antimicrobial activity as compared to the other culture conditions (Table 7.2). Similarly in case of medium F, the strain grown under the culture conditions CC3 exhibited significantly higher antimicrobial activity against two test organisms B. subtilus , S. aureus whereas it showed high antimicrobial activity against the test organism S. viridochromogens Tü 57 under the culture condition CC3. CC1 and CC3 were the conditions under which the strain demonstrated no antimicrobial attribute (Table 7.2).

Impact of different media on the antimicrobial activity of the strain CTF9 under a particular set of culture conditions

In the media B and D, the strain CTF9 gave a significantly high antimicrobial response against B. subtilus S. aureus and S. viridochromogens Tü 57 respectively, in the presence

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Cultural optimization of culture condition CC1 (Table 7.2). Strain RSF18, under the culture condition CC2 in the medium E, showed a significant antimicrobial response against B. subtilus. The other two test organisms S. aureus and S. viridochromogens Tü 57 were inhibited significantly in the medium C under the culture condition CC2 (Table 7.2). Media B, F and A succeeded in evoking antimicrobial response in the strain against the test organisms B. subtilus, S. aureus and S. viridochromogens Tü 57 respectively (Table 7.2) . Medium E and the culture condition CC4 was the combination at which the strain showed the highest antimicrobial activity against B. subtilus and S. viridochromogens Tü 57 whereas against the strain S. aureus the maximum antimicrobial activity was shown under the culture condition CC4 in the media A, D and E (Table 7.2).

DISCUSSION The nutrients and culture conditions play an important role in the onset and intensity of secondary metabolism. In order to achieve high product yields, it is a prerequisite to design a proper production medium in an efficient fermentation process (Macedo et al., 2007). There is usually a relationship between the media composition and the biosynthesis of antibiotics (Chatterjee and Vining, 1981) The role of the medium is considered in terms of the nutrients and precursors it provides to the culture (Demain et al., 1983). Six different media compositions (A, B, C, D, E, and F) along with four different sets of culture conditions (CC1, CC2, CC3 and CC4) were applied to determine the ability of the selected strains (RSF18 and CTF9) to utilize different carbon and nitrogen sources and to find out the optimal culture conditions for getting maximum yield of the antimicrobial compounds produced by them. The cultural optimization studies revealed that different media compositions and cultural conditions have significant impact on the growth and antimicrobial activity of the strains RSF18 and CTF9. The metabolic profile of the strain RSF18 has been elucidated in chapter 5, it produces cyclic thiopeptides (geninthiocin and val-geninthiocin) and the macrolide (chalcomycin). In culture optimization studies, the strain RSF18 gave maximum weight of crude extract on medium D under culture conditions CC4, (Table 7.1; Fig 7.1). Medium D contains yeast extract as nitrogen source and glucose as carbon source along with CaCl2 as a mineral salt, this is one of the simplest medium composition (consisting of only three components) among all the six media used in this study. The medium F (Fish medium,

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Table 2.31) and the medium B (SM medium, Table 2.27), yielded almost similar quantities of the crude extracts but the production level on these media is significantly low as compared to the yield observed in medium D, which depicts the restricted ability of the strain RSF18 to utilize the alternative nitrogen and carbon sources like soybeans fat, fish flour and mannitol present in media F and B. Among the other media compositions, medium A (M2 medium, Table 2.26), Medium C (Fleisch extract medium, Table 2.28), medium E (LB medium, Table 2.30), yielded minimum quantities of the crude extract (Table 7.1; Fig 7.1). On the basis of culture conditions, the maximum yield in medium D was observed on the fourth set of culture condition (CC4) i.e. pH 7.8, temperature 35oC, shaking at 110 rpm, incubation for 7 days, however the other culture conditions (CC1, CC2, and CC3)also showed similar impact and exhibited marginal variations in the weight of crude extracts obtained from a specific medium (Table 7.1; Fig 7.1). In media A, B, C, E and F, the strain RSF18 exhibited maximum growth under culture conditions CC1 and CC3, this shows the ability of the strain RSF18 to grow on a

Figure 7.1 Comparison of the effect of different media compositions and culture conditions on the yield and antimicrobial activity of the compounds produced by strain RSF18, B.S= Bacillus subtilus, S.A= Staphylococcus aureus, S.V= Streptomyces viridochromogens TU57, Wt= Weight of crude extracts

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Figure 7.2 Comparison of the effect of different media compositions and culture conditions on the yield and antimicrobial activity of the compounds produced by strain CTF9, B.S= Bacillus subtilus, S.A= Staphylococcus aureus, S.V= Streptomyces viridochromogens TU57, Wt= Weight of crude extracts wide range of temperatures i.e. 28-35oC and pH i.e. 6.5-7.8. Generally in the cultural optimization studies, the objective is usually to find out the optimal nutrients and culture conditions under which the maximum yield of the target compounds could be obtained and the production of unwanted metabolites could be diminished (Zhuang et al., 2006). Further decision for the selection of optimal nutrients and culture conditions for the strain RSF18 have been made on the basis of antimicrobial activity of the crude extracts obtained from each medium under different culture conditions against the test organisms Bacillus subtilus, Staphylococcus aureus and Streptomyces viridochromogens TU57. 40 µg of each of the corresponding crude extract from different media and culture conditions were loaded on the paper discs placed on the surface of media plates seeded with indicator micro-organisms. The variations in the antimicrobial activity depict the relative difference in the amount of active components in each of the crude extract applied. Irrespective of the significant differences in the weight of crude extracts of strain RSF18, there were marginal variations among the

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Cultural optimization antimicrobial activities of each of the crude extract (Table 7.2; Fig 7.1), showing that the proportion of the active components in each of the extract is almost similar.

The strain CTF9 producing phenyl acetic acid and β indol lacetic acid (chapter 5), in cultural optimization studies, exhibited promising growth on the culture media D, E and F, however the maximum weight of crude extract was obtained from culture medium D under the culture conditions CC2 (Table 7.1; Fig 7.2), in case of media E and F the maximum weight of crude extract was obtained under culture conditions CC1 and CC3 (Table 7.1; Fig 7.2). The similar impact of different media on the growth shows the ability of the strain CTF9 to utilize variable nitrogen sources (including yeast extract, peptone, fish flour etc) and different carbon sources added in these media. The impact of other media A, B and C on the growth of strain CTF9 has been demonstrated by the low yield of the crude extracts as compared to the media D, E and F (Table 7.1; Fig 7.2). There is also a significant impact of different culture conditions on the growth of strain CTF9. Variable quantities of the crude extracts were obtained from the same medium under different culture conditions. In the media A, B, C and D the culture conditions CC2 and CC4 showed significant impact on the growth as compared to the culture conditions CC1 and CC3, whereas in the media E and F the culture conditions CC1 and CC3 generated a significant impact on the growth as compared to the CC2 and CC4, this shows the ability of the strain CTF9 to grow on a broad range of temperatures 28- 35oC and pH between 6.5-7.8. Studies conducted on the antimicrobial activity of the crude extracts of the strain CTF9, obtained from different media and variable culture conditions, against the test organisms Bacillus subtilus, Staphylococcus aureus and Streptomyces viridochromogens TU57 in general presented almost similar activities against the test organisms, which shows that the proportion of the active components in the unit weight of the extract is more or less similar. On the basis of the results obtained in this cultural optimization study, it is possible to suggest the appropriate media and culture conditions for the strains RSF18 and CTF9. The maximum yield of the compounds produced by strain RSF18 (geninthiocin, val-geninthiocin and chalcomycin) can be obtained by cultivating the strain on medium D (yeast extract, o glucose and CaCl2) and at temperature 35 C and pH 7.8 with shaking at 110 rpm. The maximum yield of the compounds produced by strain CTF9 (phenyl acetic acid and β indol lacetic acid) can be obtained by cultivating the strain on media D, E or F, at temperature 35oC and pH 7.8.

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CHAPTER 8 MUTATIONAL ANALYSIS

Strain improvement is a vital part of process development in fermentation processes for the production of antibiotics on commercial scale. The major motivation for industrial strain development is economic, since the metabolite concentrations produced by wild strains are usually too low for the economical processes (Demain et al., 1999). Yield increases up to 100 fold or more can usually be attained through an extensive strain development program (Demain and Adrio, 2008). It provides a means by which production costs can be reduced through increases in productivity or reduction of manufacturing costs (Lee and Rho, 1999). As secondary metabolites are frequently the end result of complex, highly regulated biosynthetic process, a variety of changes in the genome may be necessary for the selection of high yielding derivatives of a wild strain. Baltz, 2001 suggested that in many cases strain improvement have been achieved using natural methods of genetic recombination, which bring together genetic elements from two different genomes into one unit to form new genotypes, however the most effective strategy is mutagenesis. A variety of chemical and physical mutagens like ethane methane sulphonates (EMS), nitrous acid, ethidium bromide, N-methyl-N'-nitro-N- nitrosoguanidine (MNNG or NTG), ultraviolet, gamma rays and X-rays are commonly used for antibiotic yield improvement in Streptomyces (Baltz and Matsushima, 1980). These mutagens induce modifications of the base sequences of DNA that result in base pair substitutions, frame shift mutations, or large deletions that go unrepaired (Kieser et al., 2000). The derivatives or mutants obtained are then subjected to screening and selection to get the strains whose characteristics are more specifically suited to the industrial fermentation process (Elander, 1967; Bos and Stadler, 1996).

The strain Streptomyces sp. RSF18 was selected to study the effect of chemical and physical mutagens on its growth and antimicrobial activity. The wild strain Streptomyces sp. RSF18 produces small colonies with 5-7 mm diameter on GYM agar, the color of substrate mycelium is yellowish with whitish spore mass (aerial mycelium) after incubation for 7-10 days. It produces three active compounds, the two cyclic thiopeptides (Geninthiocin and the new Val-geninthiocin) along with a macrolide (chalcomycin). The

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Mutational Analysis strain exhibited activity against Gram positive bacteria, and minor anti fungal activity in prescreening (Table 4.1). It was found in the preparative screening that the activity of the strain Streptomyces sp.RSF18 against gram positive bacteria is due to the presence of the identified compounds i.e. the cyclic thiopeptides (Geninthiocin and Val-geninthiocin) and the macrolide (chalcomycin) in its culture broth. For mutational analysis two chemical mutagens, ethidium bromide (10 μg ml-1, 20 μg ml-1, 50 μg ml-1 and 100 μg ml-1), N- methyl N- nitro- N- nitrosoguanidine (MNNG or NTG, 250 μg ml-1, 500 μg ml-1, 750 μg ml-1 and 1000 μg ml-1) and two physical mutagens: UV at 254 nm (for 15min, 30min, 45min and 60 min), gamma radiations (0.5 grays, 1 gray, 2 grays, 5 grays, 10 grays, 20 grays) were selected. Our major goal in this investigation was to study the effects of chemical and radiation mutagens on the yield of cyclic thiopeptides (Geninthiocin and Val-Geninthiocin) and in actual to improve the ability of the wild strain for the production of these thiopeptides. The wild strain was refreshed from stock culture and was grown on GYM agar medium for seven days to get the spore mass. In case of ethidium bromide, MNNG and UV treatments spore mass of the strain RSF18 was used, however for gamma irradiation mycelium of the wild strain was used. The mutants were selected on the basis of resistance to chloramphenicol, while the mutant selection in treatments with gamma radiation was random i.e. the derivatives which exhibited enhanced activity were selected randomly.

Antibiotics Sensitivity Pattern of the Strain Streptomyces sp. RSF18

The antibiotic sensitivity pattern of the selected strain Streptomyces sp. RSF18 was determined against different antibiotics. The wild strain Streptomyces sp. RSF18 was found to be resistant to the antibiotics, Ampicilin, Penicillin and Carbenicillin, where as Erythromycin showed intermediate sensitivity and the strain was sensitive to Novobiocin, Oxytetracycline, Chloramphenicol and Gentamycin. The inhibition zones of various antibiotics tested along with their sensitivity pattern are given in the Table 8.1. On the basis of this sensitivity pattern, the antibiotic chloramphenicol was selected as a selection marker for the isolation of mutants or derivatives of the wild strain Streptomyces sp. RSF18.

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Table 8.1 Antibiotic sensitivity pattern of wild type strain Streptomyces sp. RSF18 against various antibiotics S. No Antibiotics Disc code & Streptomyces sp. RSF18 concentration Zone size S, I, R (mm) 1 Novobiocin NV 5 μg 30 S 2 Oxytetracyclin OT 30 μg 20 S 3 Erythromycin E 15 μg 14 I 4 Ampicillin Amp 25 μg - R 5 Penicillin P 10 μg - R 6 Carbenicillin CAR 100 μg - R 7 Chloramphenicol C 30 μg 26 S 8 Gentamycin CN 10 μg 20 S

S= sensitive, zone of inhibition≥ 20mm I= intermediate, zone of inhibition≥11mm-19mm R= Resistant, zone of inhibition≥0mm- 9mm

Table 8.2 MTC of Chloramphenicol for wild type strain Streptomyces sp. RSF18 S. No Working conc. Of RSF-18 Chloramphenicol (μg/ml) Growth S, I, R. 1 1 +++ R 2 2 +++ R 3 3 ++ R 4 4 ++ I 5 5 + I 6 6 - S 7 7 - S 8 8 - S 9 9 - S 10 10 - S 11 15 - S 12 20 - S

R= Resistant, I= Intermediate, S=Sensitive (+++) = normal growth, (++) =

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Maximum Tolerable Concentration (MTC) of Chloramphenicol for the Wild Strain Streptomyces sp. RSF18

The MTC (maximum tolerable concentration) of chloramphenicol for the selected wild type strains Streptomyces sp. RSF18 was determined in order to get the appropriate chloramphenicol concentrations for mutant selection. The strain was inoculated on GYM agar plates containing different concentrations of chloramphenicol and was incubated at 28°C for 10 days, the growth of the strain was observed at different intervals. The maximum concentration that allowed the growth of the organism was considered to be the MTC (Maximum Tolerable Concentration) of the wild type strain Streptomyces sp. RSF18. The MTC for RSF18 was found to be 5 μg ml-1 (Table 8.2). The chloramphenicol concentrations 8 μg ml-1 and 10 μg ml-1 was used in the culture medium for the selection of suspected mutants of the wild strain Streptomyces sp. RSF18.

Ethidium Bromide Treatment

After the treatment of spore suspensions with different concentrations of ethidium bromide (10 μg ml-1, 20 μg ml-1, 50 μg ml-1, 100 μg ml-1 for 1 hour), 50μl of the spore suspension was spreaded on GYM agar plates containing two different concentrations of chloramphenicol (8 μg ml-1, 10 μg ml-1) and were incubated at 28˚C for two weeks. The plates were observed at regular intervals for the growth of chloramphenicol resistant mutants, after about 8-10 days few colonies appeared on the plates. The treatments with different concentrations of ethidium bromide yielded different number of chloramphenicol resistant mutant colonies (cfu), (Table 8.3). In actual 60 mutants (cfu) were obtained from all the ethidium bromide treatments on the basis of resistance against chloramphenicol. The maximum number of mutant colonies (20 cfu) was obtained from the spore suspension treated with 50 μg ml-1 ethidium bromide (Table 8.3). The chloramphenicol resistant mutant colonies obtained from ethidium bromide treatments were subjected to the evaluation of antimicrobial activity, for the final selection of the mutants with enhanced antimicrobial activities. The antimicrobial activities of these mutants were determined against the test organisms Bacillus subtillis. In treatment with 10 μg ml-1 ethidium bromide for one hour, 17 chloramphenicol resistant colonies (cfu) were obtained among them most of the mutants exhibited no change in activity, however

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6 mutants exhibited loss in activity (Table 8.3-8.4). In treatment with 20 μg ml-1 ethidium bromide for one hour, 13 chloramphenicol resistant colonies (cfu) were obtained, among them 4 mutants exhibited enhanced antimicrobial activity as compared to the parental strain, while rest of them showed reduction or loss in activity (Table 8.3-8.4). Among the 12 chloramphenicol resistant mutant colonies (cfu) obtained from 50 μg ml-1 ethidium bromide treatment only two exhibited increase in activity, while the remaining exhibited almost similar activity as compared to the parental strain (Table 8.3-8.4). In treatment with 100 μg ml-1 ethidium bromide, among the 7 chloramphenicol resistant mutant colonies (cfu) only one was found with enhanced antimicrobial activity, while others exhibited either reduction or complete loss in activity. The parental strain RSF 18 gave 14 mm zone of inhibition against Bacillus subtillis, while the mutants obtained from ethidium bromide treatments exhibited up to 20 mm zone of inhibition (Table 8.4). On the whole, out of 60 chloramphenicol resistant mutants obtained from all the ethidium bromide treatments, 9 exhibited significant increase in activity (up 40% increase in activity as compared to the parental strain in some cases), while most of the mutants showed almost similar activity and some exhibited reduced or even no activity (Table 8.4). A significant variation in the colony morphology of the selected mutants from ethidium bromide treatments was observed, usually the mutants exhibited larger colony size with greenish spore mass or aerial mycelium however some of the mutants exhibited very small compact yellowish colonies. Fig 8.1 B represents some of the selected mutants from 50 μg ml-1 ethidium bromide treatment. The enhanced antimicrobial activity of the mutants obtained by ethidium bromide treatments in comparison with the wild strain are shown in Fig 8.4 A, B.

N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) Treatment The 50μl from each of the spore suspension treated with different concentrations of MNNG (250 μg ml-1, 500 μg ml-1, 750 μg ml-1and 1000 μg ml-1for 2 hours) were plated on GYM agar plates containing chloramphenicol (8 μg ml-1, 10 μg ml-1) and were incubated at 28˚C for 2-3 weeks. The plates were observed at regular intervals for the growth of chloramphenicol resistant mutant colonies. The total number of chloramphenicol resistant mutant colonies (cfu) obtained from different MNNG treatments was 151, the maximum number of mutant colonies was obtained from the spore suspension treated with 750 μg ml-1 MNNG concentration i.e. 50 mutants (Table8.5). After the selection of chloramphenicol resistant

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Table 8.3 Number of chloramphenicol resistant mutant colonies (cfu) obtained by different ethidium bromide treatments S. No Chloramphenic Ethidium bromide treatments and the number of ol Conc. in chloramphenicol resistant colonies (cfu) obtained media (μg ml-1) 10 μg ml-1 EB 20 μg ml-1 EB 50 μg ml-1 EB 100 μg ml-1 EB 1 8 6 4 8 3 1 8 8 2 2 2 2 10 - 3 6 3 2 10 3 6 4 -

Table 8.4 Antimicrobial activity of the chloramphenicol resistant mutants obtained by ethidium bromide treatments in comparison with wild strain against Bacillus subtilus Chloramphenicol Zone of Chloramphenicol Zone of resistant mutants inhibition (mm) resistant mutants inhibition (mm) Wild Strain 14 Wild Strain 14 1.10EB/8 12 31. 20EB/10 12 2.10EB/8 14 32. 20EB/10 12 3.10EB/8 14 33. 50EB/8 10 4.10EB/8 - 34. 50EB/8 - 5.10EB/8 - 35. 50EB/8 - 6.10EB/8 15 36. 50EB/8 14 7.10EB/8 14 37. 50EB/8 18 8.10EB/8 14 38. 50EB/8 20 9.10EB/8 12 39. 50EB/8 12 10.10EB/8 14 40. 50EB/8 10 11.10EB/8 - 41. 50EB/8 - 12.10EB/8 16 42. 50EB/8 - 13.10EB/8 16 43. 50EB/10 20 14.10EB/8 14 44. 50EB/10 18 15.10EB/10 - 45. 50EB/10 14 16.10EB/10 - 46. 50EB/10 14 17.10EB/10 12 47. 50EB/10 15 18.20EB/8 - 48. 50EB/10 12 19.20EB/8 18 49. 50EB/10 12 20. 20EB/8 20 50. 50EB/10 14 21. 20EB/8 14 51. 50EB/10 - 22. 20EB/8 - 52. 50EB/10 12 23. 20EB/8 - 53. 100EB/8 12 24. 20EB/10 - 54. 100EB/8 18 25. 20EB/10 12 55. 100EB/8 - 26. 20EB/10 18 56. 100EB/8 - 27. 20EB/10 - 57. 100EB/8 14 28. 20EB/10 14 58. 100EB/10 12 29. 20EB/10 20 59. 100EB/10 - 30. 20EB/10 - 60. 100EB/10 - Key: The mutants are named according to their selection form the corresponding ethidium bromide treatment and the selection from the respective chloramphenicol concentration in GYM medium. i.e. the number indicates the mutant colony number, followed by the ethidium bromide concentration used for the treatment and /number indicates the concentration of chloramphenicol in the GYM medium e.g. 1.10EB/8, in this case 1. = mutant colony selected from the plate, 10 EB = Ethidium Bromide conc. (10μg ml-1), and /8 is the chloramphenicol conc. (8μg ml-1). 185

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mutants, the selected mutants were purified on GYM agar plates by sub culturing and were evaluated for antimicrobial activity for the final selection of mutants with enhanced antimicrobial activities. In treatment with 250 μg ml-1 of MNNG, 46 chloramphenicol resistant mutant colonies (cfu) were obtained, among them only 6 mutants exhibited increase in activity and zones of inhibition up to 18 mm were recorded as compared to the 14 mm zone of inhibition of the parental strain, while the remaining colonies exhibited no change in activity (Table 8.5-8.6). In treatment with 500 μg ml-1 of MNNG, 35 mutant colonies (cfu) were obtained, among them 10 mutants exhibited increase in activity and zones of inhibition up to 20 mm were recorded as compared to the 14 mm zone of inhibition of the parental strain, whereas most of the mutants in this treatment exhibited reduction or loss of antimicrobial activity (Table 8.5-8.6; Fig 8.3 A, B). In treatment with 750 μg ml-1 of MNNG, 50 chloramphenicol resistant mutant colonies (cfu) were recovered, among them 13 mutants exhibited increase in activity and zones of inhibition up to 22 mm were recorded as compared to the 14 mm zone of inhibition of the parental strain, while the remaining colonies exhibited reduction or complete loss in activity (Table 8.5-8.6). In treatment with 1000 μg ml-1 of MNNG, 20 chloramphenicol resistant mutant colonies (cfu) were recovered, but none of the mutant exhibited increase in antimicrobial activity rather loss of activity was exhibited by all the mutants obtained (Table 8.5-8.6). Out of 151 chloramphenicol resistant mutant colonies (cfu) recovered from different MNNG treatments, 32 exhibited significant increase in antimicrobial activity while rest of them showed almost similar, reduced or no antimicrobial activity as compared to the parental strain (Table 8.6). The mutants obtained from treatments with 500 μg ml-1and 750 μg ml-1 of MNNG exhibited maximum increase in activity i.e. up to 57% increase in antimicrobial activity (in some cases) as compared to the wild strain. (Table 8.6; Fig 8.4 C, D). The MNNG treatments also caused significant variations in the colony morphology of the selected mutants, mostly the changes in the size and texture of the colony were observed (Fig 8.1-8.2). Similarly colonies without spore mass i.e. bald colonies were obtained by 500 μg/ml MNNG treatment (Fig 8.2 E, F). The enhanced antimicrobial activity of the mutants obtained by MNNG treatment in comparison with wild strain is shown in Fig 8.4 C, D.

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Table 8.5 Number of chloramphenicol resistant mutant colonies (cfu) obtained from different MNNG treatments S. No Chloramphenicol MNNG treatments Number of chloramphenicol Conc. in media resistant mutants with (μg ml-1) 250μg ml-1 500μg ml-1 750μg ml-1 1000μg ml-1 1 8 12 5 18 10 1 8 8 9 14 4 2 10 16 13 7 6 2 10 10 8 11 -

Table 8.6 Antimicrobial activity of the chloramphenicol resistant mutants obtained by MNNG treatments in comparison with parental strain against Bacillus subtilus Chloramphenicol Zone of Chloramphenicol Zone of resistant mutants inhibition (mm) resistant mutants inhibition (mm) Parental Strain 14 Parental Strain 14 1.250MNNG/8 12 77.500MNNG/10 - 2.250MNNG/8 14 78.500MNNG/10 - 3.250MNNG/8 14 79.500MNNG/10 12 4..250MNNG/8 - 80.500MNNG/10 14 5.250MNNG/8 12 81.500MNNG/10 14 6.250MNNG/8 18 82.750MNNG/8 - 7.250MNNG/8 16 83.750MNNG/8 - 8.250MNNG/8 - 84.750MNNG/8 8 9.250MNNG/8 14 85.750MNNG/8 16 10.250MNNG/8 16 86.750MNNG/8 20 11.250MNNG/8 12 87.750MNNG/8 - 12.250MNNG/8 10 88.750MNNG/8 - 13.250MNNG/8 14 89.750MNNG/8 12 14.250MNNG/8 18 90.750MNNG/8 12 15.250MNNG/8 14 91.750MNNG/8 14 16.250MNNG/8 12 92.750MNNG/8 - 17.250MNNG/8 - 93.750MNNG/8 20 18.250MNNG/8 - 94.750MNNG/8 12

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Mutational Analysis Table 8.6 continued… 19.250MNNG/8 10 95.750MNNG/8 8 20.250MNNG/8 12 96.750MNNG/8 8 21.250MNNG/10 14 97.750MNNG/8 14 22.250MNNG/10 14 98.750MNNG/8 14 23.250MNNG/10 14 99.750MNNG/8 18 24.250MNNG/10 12 100.750MNNG/8 15 25.250MNNG/10 - 101.750MNNG/8 - 26.250MNNG/10 12 102.750MNNG/8 - 27.250MNNG/10 12 103.750MNNG/8 22 28.250MNNG/10 18 104.750MNNG/8 17 29.250MNNG/10 20 105.750MNNG/8 10 30.250MNNG/10 - 106.750MNNG/8 12 31.250MNNG/10 8 107.750MNNG/8 12 32.250MNNG/10 15 108.750MNNG/8 - 33.250MNNG/10 18 109.750MNNG/8 20 34.250MNNG/10 14 110.750MNNG/8 20 35.250MNNG/10 14 111.750MNNG/8 16 36.250MNNG/10 14 112.750MNNG/8 - 37.250MNNG/10 - 113.750MNNG/8 - 38.250MNNG/10 - 114.750MNNG/10 - 39.250MNNG/10 - 115.750MNNG/10 10 40.250MNNG/10 16 116.750MNNG/810 - 41.250MNNG/10 16 117.750MNNG/10 18 42.250MNNG/10 14 118.750MNNG/10 20 43.250MNNG/10 14 119.750MNNG/10 20 44.250MNNG/10 20 120.750MNNG/10 - 45.250MNNG/10 11 121.750MNNG/10 - 46.250MNNG/10 16 122.750MNNG/10 12 47.500MNNG/8 17 123.750MNNG/10 14 48.500MNNG/8 - 124.750MNNG/10 14

188

Mutational Analysis Table 8.6 continued…. 49.500MNNG/8 8 125.750MNNG/10 18 50.500MNNG/8 20 126.750MNNG/10 16 51.500MNNG/8 16 127.750MNNG/10 20 52.500MNNG/8 18 128.750MNNG/10 18 53.500MNNG/8 - 129.750MNNG/10 18 54.500MNNG/8 20 130.750MNNG/10 22 55.500MNNG/8 12 131.750MNNG/10 20 56.500MNNG/8 18 132.1000MNNG/8 - 57.500MNNG/8 16 133.1000MNNG/8 8 58.500MNNG/8 - 134.1000MNNG/8 - 59.500MNNG/8 10 135.1000MNNG/8 - 60.500MNNG/8 16 136.1000MNNG/8 16 61.500MNNG/10 18 137.1000MNNG/8 12 62.500MNNG/10 - 138.1000MNNG/8 12 63.500MNNG/10 - 139.1000MNNG/8 12 64.500MNNG/10 12 140.1000MNNG/8 10 65.500MNNG/10 10 141.1000MNNG/8 - 66.500MNNG/10 - 142.1000MNNG/8 - 67.500MNNG/10 - 143.1000MNNG/8 - 68.500MNNG/10 12 144.1000MNNG/8 12 69.500MNNG/10 12 145.1000MNNG/8 13 70.500MNNG/10 22 146.1000MNNG/10 16 71.500MNNG/10 20 147.1000MNNG/10 12 72.500MNNG/10 14 148.1000MNNG/10 - 73.500MNNG/10 14 149.1000MNNG/10 10 74.500MNNG/10 16 150.1000MNNG/10 - 75.500MNNG/10 20 151.1000MNNG/10 - 76.500MNNG/10 20 - - Key: The mutants are named according to their selection from the corresponding MNNG treatment and the selection from the respective chloramphenicol concentration in GYM medium i.e. the number indicates the mutant colony number, followed by the MNNG concentration used for the treatment and /number indicates the concentration of chloramphenicol in the GYM medium e.g. 1.250MNNG/8, in this case 1 = mutant colony selected from the plate, 250 MNNG= MNNG conc. (250μg ml-1), and /8 is the chloramphenicol conc. (8μg ml-1). 189

Mutational Analysis

UV irradiation The 50μl of each spore suspensions treated with UV ( at 254 nm) for different time periods i.e. 15 min, 30 min, 45 min, and 60 min, were spreaded on GYM agar plates containing chloramphenicol (8 μg ml-1, 10 μg ml-1) and were incubated at 28˚C. After 8- 10 days of incubation few chloramphenicol resistant mutant colonies were observed on the plates. The total number of chloramphenicol resistant mutant colonies (cfu) obtained by UV irradiation was 79, the maximum number of mutant colonies were obtained from the spore suspension with 15 minutes exposure time i.e. 33 cfu (Table 8.7). The selected chloramphenicol resistant mutants obtained by UV irradiation, were subjected to the evaluation of antimicrobial activity against the test organism Bacillus subtillis in comparison with the wild strain. In UV treatment with 15 minutes exposure time, 33 chloramphenicol resistant mutant colonies (cfu) were selected, among them 11 mutants exhibited enhanced antimicrobial activity and the zones of inhibition up to 20 mm were recorded as compared to the 14 mm zone of inhibition of the parantel strain, while the remaining mutants of this treatment exhibited either similar, reduced or even no activity (Table 8.7-8.8; Fig 8.3 C, D). In treatment with 30 minutes exposure time, 27 mutant colonies (cfu) were selected, among them 5 exhibited enhanced antimicrobial activity and the zones of inhibition up to 20 mm were recorded as compared to the 14 mm zone of inhibition of the parental strain, while the remaining mutant colonies exhibited reduction or even loss in activity (Table 8.7-8.8). The treatment with 45 minutes exposure time yielded 19 chloramphenicol resistant mutant colonies (cfu), however all the mutants exhibited reduced activity except one, which gave 18 mm zone of inhibition (Table 8.8). In UV treatment with 60 minutes exposure time no mutant could be recovered. Out of 79 chloramphenicol resistant mutant colonies (cfu) obtained by UV treatments, 17 showed enhanced antimicrobial activity, some mutants exhibited almost similar activity, whereas most of them showed reduced or even no activity (Table 8.8). The maximum increase in antimicrobial activity i.e. 42% increase was observed by UV treatment with 15 min exposure time (Table 8.8). The changes in the colony characteristics of the selected mutants were also observed, usually colonies with deformed or distorted shape were observed in the UV treatment for 15 min exposure time (Figure 8.1 F). The enhanced antimicrobial activity of the mutants obtained by different UV treatments in comparison with the parental strain is shown in Fig 8.4 E. 190

Mutational Analysis

Table 8.7 Number of Chloramphenicol resistant mutant colonies obtained by UV irradiation S. Chloramphenicol Conc. Number of chloramphenicol resistant mutants No in media (μg ml-1) with UV exposure at different time intervals 15 min 30 min 45 min 60 min 1 8 4 8 3 - 1 8 9 4 5 - 2 10 12 6 7 - 2 10 8 9 4 -

Table 8.8 Antimicrobial activity of chloramphenicol resistant mutants obtained by UV irradiation in comparison with parental strain against Bacillus subtilus Chloramphenicol Zone of Chloramphenicol Zone of resistant mutants inhibition resistant mutants inhibition (mm) (mm)

Wild 14 Wild 14

1.15min/8 14 41.30min/8 - 2.15min/8 12 42.30min/8 12 3.15min/8 - 43.30min/8 - 4.15min/8 14 44.30min/8 10 5.15min//8 - 45.30min/8 14 6.15min/8 16 46.30min/10 14 7.15min/8 20 47.30min/10 12 8.15min/8 12 48.30min/10 18 9.15min/8 15 49.30min/10 18 10.15min/8 8 50.30min/10 15 11.15min/8 - 51.30min/10 - 12.15min/8 18 52.30min/10 14 13.15min/8 12 53.30min/10 14 14.15min/8 - 54.30min/10 10 15.15min/10 - 55.30min/10 12 16.15min/10 - 56.30min/10 - 17.15min/10 18 57.30min/10 -

191

Mutational Analysis Table 8.8 continued… 18.15min/10 20 58.30min/10 - 19.15min/10 8 59.30min/10 8 20.15min/10 13 60.30min/10 8 21.15min/10 18 61.45min/8 - 22.15min/10 - 62.45min/8 - 23.15min/10 20 63.45min/8 - 24.15min/10 14 64.45min/8 12 25.15min/10 - 65.45min/8 12 26.15min/10 18 66.45min/8 10 27.15min/10 - 67.45min/8 - 28.15min/10 - 68.45min/8 - 29.15min/10 20 69.45min/10 14 30.15min/10 18 70.45min/10 13 31.15min/10 14 71.45min/10 18 32.15min/10 18 72.45min/10 - 33.15min/10 - 73.45min/10 - 34.15min/10 20 74.45min/10 12 35.30min/8 12 75.45min/10 10 36.30min/8 18 76.45min/10 10 37.30min/8 14 77.45min/10 - 38.30min/8 12 78.45min/10 8 39.30min/8 18 79.45min/10 - 40.30min/8 20 Key: The mutants are named according to their selection from the corresponding UV treatment and the selection from the respective chloramphenicol concentration in GYM medium. i.e. the number indicates the mutant colony number, followed by the duration of UV exposure used for the treatment and /number indicates the concentration of chloramphenicol in the GYM medium e.g. 1.15min/8, in this case 1. = mutant colony selected from the plate, 15 min = UV exposure time and /8 is the chloramphenicol conc. (8μg ml-1).

192

Mutational Analysis

Gamma Irradiation Cell mass or colonies of the wild strain were treated with gamma rays of different doses i.e. 0.5 grays, 1.0 gray, 2.0 grays, 5.0 grays 10 grays and 20 grays. The treated colonies were streaked on GYM agar plates and the mutants or derivatives were selected randomly. Total of 168 derivatives of the parental strain Streptomyces sp. RSF18 were selected randomly from all the treatments of gamma irradiation and were evaluated for antimicrobial activity (Table 8.9). In treatment with gamma irradiation of 0.5 grays 30 derivatives were selected, among them 22 were with decrease or even complete loss of activity, while 8 derivatives exhibited no change in activity (Table 8.9). In treatment of 1 grays 23 derivatives were selected among them only one mutant was found with increased activity at 11th day of incubation, while rest of the mutant colonies exhibited decreased or completely lost activity (Table 8.9). Similarly in treatment at 2 grays 30 derivatives were selected, among them 4 exhibited increased activity and all others manifested decrease or complete loss of activity (Table 8.9; Fig 8.3). Among the 30 derivatives selected from the treatment of 5 grays only one exhibited increase in activity, while rest of the selected colonies were with unchanged, decreased or lost activity. The maximum number of derivatives with enhanced activity was obtained from the treatment of 10 grays, among the 30 colonies selected in this treatment, 7 were found with increased antimicrobial activity on the 8th and 11th day of incubation, the zone of inhibition up to 20 mm was recorded as compared to the 14 mm zone of inhibition of the parental strain (Table 8.9; Fig 8.3). Similarly among the 21 derivatives selected from treatment of 20 grays only 3 exhibited increase in activity and the rest of the mutant colonies were with either decreased or completely lost activity. In all the treatments of gamma irradiation, the derivatives which exhibited variations in antimicrobial activity as compared to the wild strain were selected, while those which exhibited similar activity in comparison with parental strain were ignored. On the whole 50 mutants were finally selected, among them 15 exhibited enhanced activity i.e. about 42% increase in activity as compared to the parental strain, while rest of them showed decreased or complete loss of activity (Table 8.9). The enhanced antimicrobial activities of the selected mutants obtained with gamma irradiation relative to that of parental strain are shown in Fig 8.4 F.

193

Mutational Analysis

Table 8.9 Antimicrobial activity at different time intervals of randomly selected derivatives of RSF18 after treatment with gamma irradiation of different doses S. No. Mutants/derivatives Antimicrobial activity against Bacillus subtilus of RSF18 (zone of inhibition in mm) 5th day 8th day 11th day Streptomyces sp. RSF18 + 14 14 (Parental strain) Selected derivatives by treatment with gamma rays at 0.5 grays 1 1/0.5 grays + 14 - 2 2/0.5 grays + 14 - 3 3/0.5 grays + 14 12 4 4/0.5 grays + 14 14 5 5/0.5 grays - 10 12 6 6/0.5 grays - 10 10 7 7/0.5 grays - 8 9 8 8/0.5 grays - 8 10 9 9/0.5 grays - 8 10 10 10/0.5 grays - 9 9 11 11/0.5 grays - 10 10 12 12/0.5 grays - 9 8 13 13/0.5 grays - 14 16 14 14/0.5 grays - - - 15 15/0.5 grays - 8 14 16 16/0.5 grays - 10 13 17 17/0.5 grays - 12 14 18 18/0.5 grays - 11 10 19 19/0.5 grays - 9 10 20 20/0.5 grays - 14 15 21 21/0.5 grays - 14 15 22 22/0.5 grays - 11 13 23 23/0.5 grays - 14 14 24 24/0.5 grays - 14 14

194

Mutational Analysis

25 25/0.5 grays - 11 15 26 26/0.5 grays - - 6 27 27/0.5 grays - - 12 28 28/0.5 grays - - 6 29 29/0.5 grays - - 14 30 30/0.5 grays - - 13 Selected derivatives by treatment with gamma rays at 1 grays 31 1/1.0 grays - - - 32 2/1.0 grays - - 12 33 3/1.0 grays - - 6 34 4/1.0 grays - - 13 35 5/1.0 grays - - 16 36 6/1.0 grays - - 14 37 7/1.0 grays - - - 38 8/1.0 grays - - 10 39 9/1.0 grays - 10 15 40 10/1.0 grays - - 15 41 11/1.0 grays - 11 16 42 12/1.0 grays - - - 43 13/1.0 grays - 15 19 44 14/1.0 grays - - - 45 15/1.0 grays - - - 46 16/1.0 grays - 10 - 47 17/1.0 grays - 12 14 48 18/1.0 grays - 11 9 49 19/1.0 grays - 10 - 50 20/1.0 grays - - - 51 21/1.0 grays - 10 12 52 22/1.0 grays - 11 16 53 23/1.0 grays - 14 14

195

Mutational Analysis

Selected derivatives by treatment with gamma rays at 2 grays 54 1/2.0 grays - 10 16 55 2/2.0 grays - 11 20 56 3/2.0 grays - - 12 57 4/2.0 grays - - 16 58 5/2.0 grays - - 17 59 6/2.0 grays - - 10 60 7/2.0 grays + 11 16 61 8/2.0 grays + 12 15 62 9/2.0 grays - 10 15 63 10/2.0 grays - - - 64 11/2.0 grays - - - 65 12/2.0 grays - - - 66 13/2.0 grays - - 8 67 14/2.0 grays - 10 8 68 15/2.0 grays - 9 - 69 16/2.0 grays - 9 12 70 17/2.0 grays - 16 18 71 18/2.0 grays - 8 - 72 19/2.0 grays - 12 10 73 20/2.0 grays - - - 74 21/2.0 grays - - 8 75 22/2.0 grays - 12 8 76 23/2.0 grays - 13 10 77 24/2.0 grays - 9 - 78 25/2.0 grays - 10 11 79 26/2.0 grays - 19 15 80 27/2.0 grays - 20 15 81 28/2.0 grays - 18 15 82 29/2.0 grays - - 10

196

Mutational Analysis

83 30/2.0 grays - - 9 84 31/2.0 grays + - 13 Selected derivatives by treatment with gamma rays at 5 grays 85 1/5.0 grays - - 12 86 2/5.0 grays + - 12 87 3/5.0 grays + 13 16 88 4/5.0 grays - - 14 89 5/5.0 grays + 9 11 90 6/5.0 grays + 9 10 91 7/5.0 grays - 11 11 92 8/5.0 grays - - 9 93 9/5.0 grays - 15 18 94 10/5.0 grays - 15 14 95 11/5.0 grays + 16 16 96 12/5.0 grays - 9 8 97 13/5.0 grays - 12 9 98 14/5.0 grays - 12 12 99 15/5.0 grays - 10 - 100 16/5.0 grays - 12 12 101 17/5.0 grays ++ - 15 102 18/5.0 grays + 11 15 103 19/5.0 grays +++ 11 12 104 20/5.0 grays - - 15 105 21/5.0 grays - 9 15 106 22/5.0 grays - 9 - 107 23/5.0 grays - - - 108 24/5.0 grays - - 8 109 25/5.0 grays - 8 8 110 26/5.0 grays - 8 8 111 27/5.0 grays - 14 -

197

Mutational Analysis

112 28/5.0 grays - 10 - 113 29/5.0 grays - 12 14 114 30/5.0 grays - 12 14 115 31/5.0 grays - 12 14 Selected derivatives by treatment with gamma rays at 10 grays 116 1/10.0 grays - - - 117 2/10.0 grays - - - 118 3/10.0 grays - - - 119 4/10.0 grays - 9 14 120 5/10.0 grays - 8 12 121 6/10.0 grays + 12 16 122 7/10.0 grays + 11 10 123 8/10.0 grays - 10 11 124 9/10.0 grays - - - 125 10/10.0 grays - 10 10 126 11/10.0 grays - 10 11 127 12/10.0 grays - - - 128 13/10.0 grays - - - 129 14/10.0 grays - - 10 130 15/10.0 grays - - 12 131 16/10.0 grays - 13 14 132 17/10.0 grays - 12 14 133 18/10.0 grays - 18 14 134 19/10.0 grays - 20 19 135 20/10.0 grays - - 18 136 21/10.0 grays - 7 10 137 22/10.0 grays - - 18 138 23/10.0 grays - - 8 139 24/10.0 grays - 9 8 140 25/10.0 grays + 18 17 141 26/10.0 grays - 12 10

198

Mutational Analysis

142 27/10.0 grays + 20 - 143 28/10.0 grays - 19 18 144 29/10.0 grays - 20 18 145 30/10.0 grays - 14 - 146 31/10.0 grays - 13 13 Selected derivatives by treatment with gamma rays at 20 grays 147 1/20.0 grays - 18 8 148 2/20.0 grays - 12 - 149 3/20.0 grays - 12 - 150 4/20.0 grays - 12 - 151 5/20.0 grays ++ - 10 152 6/20.0 grays - 14 10 153 7/20.0 grays - - 9 154 8/20.0 grays - 20 19 155 9/20.0 grays - 9 - 156 10/20.0 grays - 11 - 157 11/20.0 grays 14 14 158 12/20.0 grays - 16 14 159 13/20.0 grays - 11 - 160 14/20.0 grays - 12 11 161 15/20.0 grays - 11 11 162 16/20.0 grays - - - 163 17/20.0 grays - 18 10 164 18/20.0 grays - 14 - 165 19/20.0 grays - 14 - 166 20/20.0 grays - - - 167 21/20.0 grays - 14 10 168 22/20.0 grays - 12 -

(-) no activity, (+) minor activity, (++, +++) significant activity, The mutants are named according to their selection from the corresponding gamma radiation treatment and the selection from the respective chloramphenicol concentration in GYM medium i.e. the number indicates the mutant colony number, followed by the irradiation with gamma rays and /number indicates the concentration of chloramphenicol in the GYM medium e.g. 1/0.5grays, in this case 1. = mutant colony selected from the plate, 0.5 grays = dose of gamma rays.

199

Mutational Analysis

A B

C D

E F

Fig 8.1 Selected chloramphenicol resistant mutants of wild strain RSF18 obtained by different mutagenic treatments exhibiting variations in morphologic characters, A- morphologic pattern of wild strain RSF18 on -1 GYM medium, B- mutants obtained by 50μg ml ethidium bromide treatments, C, D, E- chloramphenicol resistant mutants obtained by MNNG treatments, F- chloramphenicol resistant mutants obtained by UV irradiation at 254nm for 15 min

200

Mutational Analysis

A B

C D

E F Fig 8.2 Chloramphenicol resistant mutants of wild strain RSF18 exhibiting variations in colony characteristics obtained by treatments with different concentrations of MNNG. A, B, C D = mutants -1 -1 obtained by 750μg ml MNNG treatment, E, F = bald mutants obtained from 500μg ml MNNG treatment.

201

Mutational Analysis

A B

C D

E F

Fig 8.3 Variations in antimicrobial activity of the Chloramphenicol resistant mutants of parental strain Streptomyces sp. RSF18 obtained by different mutagenic treatments. A, B = antimicrobial activity pattern of the mutants obtained by 500μg ml-1MNNG treatment, C, D = antimicrobial activity pattern of the mutants obtained by UV exposure for 15 min, E = antimicrobial activity pattern (at 11th day of incubation) of the mutants obtained by irradiation with gamma radiations at 2 grays, F = antimicrobial activity pattern of the mutants (at 11th day of incubation) obtained by irradiation with gamma rays at 10 grays.

202

Mutational Analysis

Wild Wild

A B

Wild Wild

C D

Wild Wild

E F

Figure 8.4 Enhanced antimicrobial activity of finally selected mutants against Bacillus subtilus in comparison with the wild strain RSF18, A, B= mutants selected from ethidium bromide treatments exhibiting enhanced antimicrobial activity in comparison with parental strain, C, D= mutants selected from MNNG treatments exhibiting enhanced antimicrobial activity in comparison with parental strain, E= mutants selected from treatment with UV (at 254nm) exhibiting enhanced antimicrobial activity in comparison with wild strain, D= mutants selected from gamma irradiation exhibiting enhanced antimicrobial activity in comparison to parental strain.

203

Mutational Analysis

Discussion

Mutagenesis has been a very important strategy for strain improvement, it has been extensively applied to enhance the product yield, to inhibit the biosynthesis of unwanted metabolites in a production strain and to get the derivatives of a production strain that can grow on cheaper carbon and nitrogen sources or cost effective culture media (Baltz, 1986). The results presented here show the effects of commonly used chemical mutagens like ethidium bromide, MNNG and physical mutagens like UV and ionizing radiation i.e. gamma rays on the growth behavior and antimicrobial activity of the wild strain Streptomyces sp. RSF18. As the metabolic profile of the wild strain is already established and the activity of the compounds produced by this strain is known i.e. the compounds (Geninthiocin, Val- geninthiocin and Chalcomycin) are active against Gram positive bacteria, so the change in activity against gram positive bacteria was considered as indication of the change in the level of production of these compounds. The mutagenic treatments of Streptomyces sp. RSF18 with chemical and radiation mutagens exhibited significant variations in antimicrobial activity of the mutants against Gram positive bacteria and yielded a good number of mutants with enhanced activity. Two different criteria were adopted for the selection of mutants i.e. random selection and selection on the basis of resistance to a selection marker (resistance to antibiotic chloramphenicol), for the sake of the comparison of procedures adopted for selection. The mutants were selected randomly in case of gamma irradiation where as they were selected for chloramphenicol resistance in case of ethidium bromide, MNNG and UV treatments. It was observed that the wild strain can tolerate maximum concentration of chloramphenicol up to 5μg ml-1 (Table 8.2), so for the selection of mutants in case of ethidium bromide, MNNG and UV treatments almost two fold higher concentrations of chloramphenicol (8μg ml-1 and 10μg ml-1) were used.

Spores of the Streptomyces sp. RSF18 were treated with four different concentrations i.e. 10μg ml-1, 20μg ml-1, 50μg ml-1 and 100μg ml-1 of ethidium bromide. The maximum number of chloramphenicol resistant mutant colonies (50 mutants) was obtained from the spores treated with 50μg ml-1 ethidium bromide (Table 8.3; Fig 8.5). The ethidium bromide treatments generally resulted in the reduction or loss of antimicrobial activity, as the selected mutants mostly exhibited reduction or loss in activity and very few showed enhanced activity. It was observed that the mutants obtained from lower concentrations of ethidium

204

Mutational Analysis bromide 10μg ml-1, exhibited almost similar activity as compared to the parental strain however complete loss of activity was also observed with this treatment. The significant variations in activity (increase or decrease) were exhibited by the mutants obtained with 20μg ml-1 and 50μg ml-1 ethidium bromide treatments while the mutants obtained from 100μg ml-1 ethidium bromide concentration exhibited mostly reduction or loss in activity (Table 8.4). Among the 60 selected chloramphenicol resistant mutant colonies (cfu) from all the ethidium bromide treatments, only 9 shows significantly enhanced antimicrobial activity as compared to the parental strain. Hence on the basis of results obtained it can be concluded that the ethidium bromide concentrations 20μg ml-1 and 50μg ml-1 are more effective for yielding mutants of the Streptomyces sp. RSF18 with enhanced antimicrobial activity. The selected mutants also exhibited marked variations in colony characteristics, colonies with large size and greenish spore masses were observed (Fig 8.1 B).

Figure 8.5 Number of chloremphenicol resistant mutant colonies (cfu) obtained by treatment with different concentrations of ehtidium bromide

In case of MNNG treatments the spores of the wild strain were treated with four different concentrations i.e. 250μg ml-1, 500μg ml-1, 750μg ml-1 and 1000μg ml-1. In manifestation of antimicrobial activity, the mutants obtained by MNNG treatments exhibited significant variations including increase, decrease or complete loss in antimicrobial activity. Total of 151 chloramphenicol resistant mutant colonies (cfu) were selected from all the four MNNG treatments (Table 8.5). On the basis of extensive screening for variations in antimicrobial

205

Mutational Analysis activity, 17 chloramphenicol resistant mutants exhibited enhanced antimicrobial activity against Bacillus subtillis. The maximum number of chloramphenicol resistant mutant colonies (50 mutants) was obtained from the spores treated with 750μg ml-1 of MNNG. In general it was observed that the lower concentrations of MNNG i.e. 250μg ml-1 had poor effect on the antimicrobial activity as the chloramphenicol resistant mutants obtained with this treatment exhibited almost similar activity as that of parental strain, however few mutants with enhanced activity were also recovered (Table 8.6). The treatments of 500μg ml- 1 and 750μg ml-1 MNNG yielded mutant colonies exhibiting significant changes (increase or decrease) in antimicrobial activity. The high concentration (1000μg/ml) of MNNG had detrimental effects on Streptomyces sp. RSF18. This concentration yielded few chloramphenicol resistant mutants which exhibited either decrease or complete loss of antimicrobial activity. The mutants obtained with MNNG treatments also exhibited significant differences in colony characteristics including changes in the size, pigmentation and spore formation (Fig 8.1 C, D, E). Bald colonies were obtained from the spores treated with 500μg ml-1 of MNNG (Fig 8.2 E, F).

MNNG is very effective mutagen when used under appropriate conditions, it yields the highest mutant frequencies than any other mutagen at low lethality (Kieser et al., 2000). MNNG causes GC-AT transitions and to a small extent deletions and frame shift mutations, according to one possible explanation the compound is easily decomposed in vivo and in acidic conditions nitrous acid is formed, while diazomethane a strongly methylating agent is formed under alkaline conditions (Hidaka et al., 1978). In this study the criteria for mutant selection in case of (Ethidium Bromide, MNNG and UV treatments) was the enhanced resistance to chloramphenicol, so it is possible that the treatment with lower concentration of the mutagen might have caused the resistance to chloramphenicol but the chances of occurrence of multiple mutations that may lead to the change in antimicrobial activity were less, hence the mutants obtained with mutagenic treatment at lower concentrations exhibited minor variations in the antimicrobial activity. In treatments with higher concentrations chances of multiple changes may be high, which ultimately caused resistance to chloramphenicol and also affected the antimicrobial activity of the mutants obtained from these treatments.

The treatments with short wave length UV (UV at 254 nm) at different time intervals yielded

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Mutational Analysis

Figure 8.6 Number of chloremphenicol resistant mutant colonies (cfu) obtained by treatment with different concentrations of MNNG

79 chloramphenicol resistant mutant colonies (cfu). On the basis of extensive screening of chloramphenicol resistant mutants for antimicrobial activity, 12 mutants were obtained with significant increase in activity against Gram positive bacteria (Table 8.5-8.6). The exposure time of 15 min is more effective as maximum number of chloramphenicol resistant mutants (30) was obtained from this treatment. There was a gradual decrease in the number of chloramphenicol resistant mutants obtained from long time UV exposures i.e. 30 min, 45 min and 60 min (Table 8.6). The maximum increase in antimicrobial activity was observed in the mutants obtained from 15 min and 30 min exposure times. UV treatment also cause changes in colony characteristics i.e. colonies with larger size, flate in elevation and with irregular margins were obtained (Fig 8.1 F). Among the physical mutagens UV is a very convenient and comparatively safe mutagen, but it may fail to be effective in some species, either because a strain lack the error prone repair system needed to convert premutagenic change in to a heritable mutation or for unknown reasons, as in case of Streptomyces fradiae (Baltz, 1986). The most important products of UV action are dimmers i.e thymine-thymine, thymine-cytocine, cytocine-cytocine dimmers, formed between the adjacent pyrimidines or between the pyrimidines of complimentary strands and results in cross linking. UV radiations also induce transitions of GC-AT, transversions, frameshift mutations and deletions (Kieser et al., 2000).

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Mutational Analysis

Figure 8.7 Number of chloremphenicol resistant mutant colonies (cfu) obtained with UV irradiation at different time intervals.

The treatment with gamma rays was given to the mycelium of the wild strain Streptomyces sp. RSF18 instead of the spores. Almost 168 derivatives were randomly selected on the basis of the variations in antimicrobial activity against gram positive bacteria. The derivatives obtained by treatments with gamma rays were slow in growth, hence the antimicrobial activity was determined at different times after incubation i.e. at 5th, 8th and 11th days of incubation. In general most of the derivatives exhibited almost similar activity as compared to the parental strain, however increase, decrease and complete loss of activity was also observed. A significant increase in antimicrobial activity was exhibited by 15 mutants from this group. The irradiation at 10 grays and 20 grays intensity were found to be most effective as maximum derivatives with enhanced activity were obtained from these treatments (Table 8.9). It seems important to mention here that no significant change in colony characteristics was observed in the mutants obtained with gamma irradiation. Gamma rays act by causing the ionization of the medium through which they pass and cause single and double strand breaks with a significantly higher probability, which leads to the structural changes such as translocations, inversions or similar chromosomal mutations.

The %age increase in antimicrobial activity for each of the mutagenic treatment was calculated by determining the increase in the size of zone of inhibition exhibited by mutants

208

Mutational Analysis

Figure 8.8 %age increase in the antimicrobial activity of the parental strain RSF18 by different mutagenic treatments (EB i.e. ethidium bromide, 1= 10μg ml-1, 2= 20μg ml-1, 3= 50μg ml-1, 4= 100μg ml-1, MNNG, 1= 250μg ml-1, 2=500μg ml-1, 3= 750μg ml-1, 4= 1000μg ml-1), (UV, 1= 15 min, 2= 30 min, 3= 45 min, 4= 60 min), (GR i.e. gamma rays, 1= 0.5 grays, 2= 1 grays, 3= 2 grays, 4= 5 grays, 5= 10 grays, 6= 20 grays). of a particular mutagenic treatment. Usually the zones of inhibition up to 18 mm 20 mm or 22 mm were exhibited by the mutants of different mutagenic treatments, thus increases up to 4 mm, 6 mm or 8 mm in the size of the inhibition zone were recorded as compared to the 14 mm zone of the wild strain, which resulted into 28 %, 42% and 57% increase respectively. The maximum increase in antimicrobial activity i.e. 57% was recorded in treatment with 750μg/ml MNNG (Fig 8.8). The other mutagenic treatments i.e. 20μg/ml and 50μg/ml ethidium bromide, 500μg/ml MNNG, UV exposure for 15 minutes and gamma irradiation at 10 grays causes 42% increase in antimicrobial activity of the respective mutants as compared to the wild strain (Fig 8.8). No doubt mutants with reduced activity or even no activity were also obtained in all the mutagenic treatments, however the treatments, 10μg/ml ethidium bromide, 250μg/ml, MNNG, 45 min and 60 minutes exposure to UV at 254nm, and gamma irradiation at 0.5 grays, 1 grays and 2 grays, either caused no effect, reduction or even sometime 100 % decrease in antimicrobial activity of the mutants as compared to the wild strain.

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Discussion

CHAPTER 9 DISCUSSION Natural products continue to be a useful source of new leads for the pharmaceutical industry. The usual definition of natural products in the widest sense emphasizes that they are the chemical compounds isolated from diverse living things. They are usually the secondary metabolites of living organisms with low molecular weight (MW<3000), and are characteristic mainly to some specific, distinct types of organisms (Berdy, 2005). Natural products being the secondary metabolites may be produced by almost all types of living forms, however various actinomycetales, first of all the Streptomyces species and filamentous fungi, and to a lesser extent several bacterial species are the most noteworthy producers both in respect of numbers, versatility and diversity of structures of the produced metabolites. The search for and exploitation of the microbial sources for bioactive natural products have been the major objective of the biotechnology. Actinomycetes are prolific producers of natural products and one strategy to increase the possibility of discovering novel chemical entities is to screen actinomycetes considered `rare'' in the environment and previously under-represented in natural product screening collections (Blunt et al., 2003). Streptomyces is the most extensively studied genus of actinomycetes, the members of which have been reported as the major producers of bioactive natural products. Although members of Streptomyces have long been extensively screened for bioactive compounds, rare species of this genus are expected to contain undiscovered isolates and metabolic products (Vickers et al., 1984; Goodfellow and Williams 1986; Bull et al., 2000). Irrespective of the world wide exploitation of Streptomyces for bioactive secondary metabolites, these organisms has been least studied in this area (Pakistan) and previously almost there are no reports about the biodiversity, antagonistic behavior and nature of the metabolites produced by the Streptomyces flora of this ecological part of the world. Keeping in view the fact that the streptomycetes present under different climatic condition and in unexplored ecological niche may produce unique or interesting metabolites (Ball et al., 1989; Wachinger et al., 1989), the Streptomyces flora of 30 soil samples collected from different localities, including saline farming lands in the province Punjab and northern areas of Pakistan, were screened for their potential as a source of

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Discussion antibiotics. The Streptomyces strains were isolated on selective media by applying different physical and chemical treatments to soil samples and stringent culture conditions as suggested by Hayakawa et al. (2004). Out of 110 selected bioactive Streptomyces strains the isolates RSF17, RSF18, RSF23 (rose field isolates), BG5 isolated from the soil of botanical garden, CRF1, CRF2, CRF11, CRF14, CRF17 (corn field isolates), SCF18, SCF25, SCF31 (sugar cane field isolates), CTF9, CTF13, CTF14, CTF15, CTF20, CTF23, CTF24 and CTF25 (cotton field isolates) were considered as the representative strains and were studied in detail. Streptomyces can be easily distinguished from all other bacterial groups by their unique growth pattern, hard and embedded colonies, diffused pigments and the color of substrate and aerial mycelia (Anderson and Wellington, 2001). The physiological characterization especially criteria employed in International Streptomyces Project (ISP) i.e. production of melanin and utilization of different sugars as carbon source (Shirling and Gottlieb, 1966), and the results of some additional physiological characteristics like, determination of growth temperature range, utilization of organic acids, utilization of oxalates, hydrolysis of urea and hemolysis test (Lechevalier, 1989), provided further confirmation for the identification of these isolates up to the genus level. Accurate identification of the producing microorganisms in natural product research is of great importance because antibacterial and antifungal compounds production, and synthesis of extracellular metabolites, are usually strain-specific and may be associated with certain species and subspecies of the microorganisms (Pinchuk et al., 2002). Along with the morphological, biochemical and physiological characterization, the 16s rRNA gene sequencing being the most reliable technique was adopted for confirming the exact taxonomic status of the selected Streptomyces strains. In general, the comparison of the 16S rRNA gene sequences allows differentiation between organisms at the genus level across all major phyla of bacteria, in addition to classifying strains at species and subspecies level (Woese et al., 1985). The sequences obtained were rechecked manually for errors and the data was refined for BLAST analysis. The highest homology matches returned from BLAST searches confirmed that all the isolates are different species of the genus Streptomyces. In comparison of the strains among themselves on the basis of biochemical and physiological characteristics, the data was arranged in the form of numerals for numerical

211

Discussion analysis and was compared by the computer software MINITAB version 11. The resulting Dandrogram shows that the strains arranged into two clusters (phenons with 42% similarity), a cluster of the isolates CRF14, CTF23 and CTF24 which are 61.5% similar to each other and a cluster of the remaining seventeen isolates which also exhibit 61.5% similarity among themselves. In numerical taxonomic analysis of the Streptomyces, the phenons formed at 80% similarity often are equivalent to specie, while the phenons formed at 60% are usually considered equivalent to the same genus (Kampfer et al., 1991). As the members in each of the cluster are more than 60% similar to each other, so they can be considered to be belonging to the same genus. Generally it was observed that the isolates with the same origin are closely related to each other for example the rose field isolates RSF17, RSF18 and RSF23 exhibit maximum similarity with each other (Fig 9.1). The isolate BG5 (isolated from botanical garden) exhibited maximum similarity with the isolate CTF9 (cotton field isolate), similarly the corn field isolates CRF1, CRF2, CRF11, and CRF17 exhibited similarity with each other and also with different cotton field isolates (Fig 9.1). The sugarcane field isolates SCF18, SCF25 and SCF31 exhibited similarity among them and also showed close similarity with the corn field isolate CRF17 and Cotton field isolates CTF20 and CTF25. Additionally the comparison of the 16s rRNA gene sequences of the isolates with each other shows the genetic similarity of the isolates among themselves. The Dandrogram (Fig 9.2) obtained using 16s rRNA gene sequences of the selected Streptomyces strains show the clustering by unweighted pair group method with arithmetic means (UPGMA). The isolates rearranged and joined to form clusters of similar strains, basically four clusters were obtained on the basis of genetic relatedness and the results are in accordance with the similarity found among the isolates by physiological characterization. The cluster of the isolates RSF17, RSF18 and SCF18 is genetically closer to the cluster of the isolates CTF13, CTF14, CRF2 and CTF15 (Fig 9.2). Similarly the cluster of the isolate CTF9, CTF25, and SCF25 is similar to the isolate CTF20 and RSF23 and the cluster of the isolates BG5, CRF17 and SCF23 exhibited close genetic similarity to the isolate CRF1 (Fig 9.2).

In screening for bioactive metabolites, the useful procedures involve biological screening followed by bioassay guided isolation of the respective compounds (Taddei et al., 2006).

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Discussion

Numbers 1-20 (1=RSF17,2=RSF18, 3=RSF23, 4=BG5, 5=CRF1, 6=CRF2, 7=CRF11, 8=CRF14, 9=CRF17, 10=SCF18, 11=SCF25, 12=SCF31, 13=CTF9, 14=CTF13, 15=CTF14, 16=CTF15, 17=CTF20, 18=CTF23, 19=CTF24, 20=CTF25)

Figure 9.1 Similarity among the isolates on the basis of different physiological characteristics including production of melanin, utilization of different sugars as carbon source, utilization of organic acids, utilization of oxalates, hydrolysis of urea and hemolysis, the cluster analysis was performed by MINITAB version11.

Fig: 9.2 Dendrogram obtained using 16s rRNA gene sequences of the selected Streptomyces strains showing clustering by the unweighted pair group method with arithmetic means (UPGMA).

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Discussion

The biological screening of the selected Streptomyces isolates against a panel of nine test organisms including Gram positive bacteria, Gram negative bacteria, fungi and microalgae by disc diffusion method, additionally the cytotoxicity analysis performed by brine shrimp (Artimia salina) microwell cytotoxicity assay, provided a wide ranging information about the antimicrobial activity and gave some clues about the nature of the metabolites produced by these isolates. During the selection of a competent strain from a strain collection, many potentially interesting isolates might be excluded due to inadequate techniques, too high selectivities or just because of missing tests (Taddei et al., 2006), we therefore used a so-called horizontal screening, i.e. a combination of tests with low selectivity covering a broad range of antibacterial, antifungal, phycotoxic and cytotoxic activities (Laatsch 2000). It was assumed that the Streptomyces isolate producing highly active crude extracts with broad range of antimicrobial activities and low cytotoxic effects may be considered as interesting stains, because generally it is assumed that the strain exhibiting high cytotoxity may be producing some trivial compounds like actinomycins etc or at least the compounds which may not be useful to be used as antibiotics (Laatsch 2000). Each of the isolate exhibited a specific activity pattern e.g. strains RSF17, RSF18 and RSF23 the rose field isolates exhibited almost similar pattern of activity (Fig 9.3), which may indicate that these isolates and all others (in strain collection) coming from the same origin exhibit broad spectrum activities and might be producing similar types of metabolites. Further decision for the selection of a competent strain from this group can be made from cytotoxicity results, the isolate RSF23 exhibit high cytotoxicity, almost 93% mortality (Fig 9.4), which indicate the compounds produced by this isolate may not be very interesting due to their cytotoxic effects, however the isolates RSF17 and RSF18 exhibited low cytotoxicity (15% and 13% mortality respectively) and can be selected for preparative screening. The isolate BG5 yielded highly active crude extract, its broad spectrum activity (Fig 9.3) and low cytotoxicity (Fig 9.4) predicted that it could be an interesting strain for further studies. Among the isolates obtained from the soil of corn fields, the strains CRF1 and CRF2 show almost similar type of activity pattern, however the strains CRF11, CRF14, and CRF17 exhibited broad spectrum activity (Fig 9.3), the cytotoxicity results of these strains showed that the strain CRF11 is highly cytotoxic (Fig 9.4) (almost 97% mortality

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Discussion

25 S.A 20 B.S Tu 57 15 E.C

10 C.A

M.M Zoneof inhibition (mm) 5 C.V C.S 0 S.S RSF RSF RSF BG CRF CRF CRF CRF CRF SCF SCF SCF CTF CTF CTF CTF CTF CTF CTF CTF 17 18 23 5 1 2 11 14 17 18 25 31 9 13 14 15 20 23 24 25 Streptomyces isolates

(S.A= Staphylococcus aureus, B.S= Bacillus subtilus, Tu 57= Streptomyces viridochromogens Tu 57, E.C= E coli, C.A= Candida albicans, M.M= Mucor mehimi, C.V= Chlorella vulgaris, C.S= Chlorella sorokiana, S.S= Scendesmus subspicatus) Figure 9.3 Antimicrobial activity pattern of the selected Streptomyces isolates

120

100

%age mortality %age 40

0 RSF RSF RSF BG CRF CRF CRF CRF CRF SCF SCF SCF CTF CTF CTF CTF CTF CTF CTF CTF 17 18 23 5 1 2 11 14 17 18 25 31 9 13 14 15 20 23 24 25 Streptomyces isolates

Figure 9.4 Cytotoxicity of the selected Streptomyces isolates determined by brine shrimp (Artimia salina) microwell cytotoxity assay

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Discussion was observed) so it can be excluded from the race of selection of a competent strain. Similarly the antimicrobial activity pattern of the Streptomyces isolates from sugar cane field i.e. SCF18, SCF25, SCF31 (Fig 9.3) suggested that the isolate SCF25 produce more active crude extract and seems to be an interesting strain. The Streptomyces isolates CTF9, CTF13, CTF14, CTF15, CTF20, CTF23, CTF24, CTF25 are the next major group isolated from the soil of cotton field , their antimicrobial activity pattern and cytotoxicity analysis (Fig 9.3- 9.4) suggested that the isolates CTF9, CTF13, CTF15 and CTF20 could be selected for further studies. In chemical screening the techniques as simple as TLC is usually used, but now a days preference is being given to HPLC linked to different detectors (hyphenated techniques). Instruments with HPLC coupled to UV (LC-UV) have been extensively used in chemical screening, however the combination of HPLC-MS is the next major advance in chemical screening. High-performance liquid chromatography (HPLC) coupled to electrospray ionization tandem mass spectrometry (ESI-MS/MS) plays an increasing role in natural product analysis, since it permits the fast screening of crude biological extracts for detailed information about metabolic profiles, with a minimum amount of material (Bringmann et al., 1999; Pelillo et al., 2004). In addition to biological screening, the evaluation of the isolates by chemical screening provided comprehensive information of the secondary metabolites produced by these Streptomyces strains. The analysis of the crude extracts of the individual strains on TLC exhibited different UV (at 254 and 366 nm) absorbing fractions and the marked patterns of colour reactions with staining reagents (Eherlich’s reagent and Anisaldehyde/H2SO4). The analysis of each of the crude extract by HPLC-MS/MS provided basically two types of information, firstly the possible number of fractions in a crude extract as it was seen in the total ion chromatogram of an extract (Fig 9.5), secondly the mass of each of the fraction in a crude extract. The chemical screening results demonstrated that each isolate presented a unique pattern of secondary metabolites independently of their origin. Based upon the secondary metabolite patterns obtained by TLC as colour reaction or UV absorbing spots along with the HPLC-MS/MS analysis, it was possible to compare an individual strain with the related strains in the strain collection and to decide whether it should be selected for further studies or not. The final decision for the selection of a strain for further study was

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Discussion

Table 9.1 Summary of the selected strains for preparative screening on the basis of pre screening results and specific criteria of selection S. No. Isolate Name Criteria of selection

1 RSF18 Streptomyces  Activity against Gram positive, Gram sp.RSF18 negative bacteria  Antifungal activity  Low cytotoxicity (15% mortality of the shrimp larvae)  Unique blue spots on TLC  Positive peptide test  Metabolites with small number of hits in AntiBase 2 CRF17 Streptomyces  Representative member of the corn pulcher field isolates  Antibacterial and antifungal activity  Strong antialgal activity  Unique TLC bands  Metabolites with small number of hits in AntiBase 3 BG5 Streptomyces  Broad spectrum activity matensis  Low cytotoxicity (28% mortality of the shrimp larvae)  Unique yellowish spots on TLC  Large number of metabolites in HPLC- MS analysis 4 CTF9 Streptomyces  Strong antifungal and anti algal malachitofuscus activity  Low cytotoxicity (11% mortality of the shrimp larvae)  Unique yellowish spots on TLC  Large number of metabolites in HPLC- MS analysis 5 CTF15 Streptomyces  Activity against Gram positive bacteria griseoincarnatus  High cytotoxicity (91.2% mortality of the shrimp larvae)  Large size UV absorbing greenish yellow spots on TLC  Large number of metabolites in HPLC- MS analysis 6 SCF25 Streptomyces  Broad spectrum activity macrosporeus  High cytotoxicity(91.4% mortality of the shrimp larvae)  Unique reddish and yellow spots on TLC  Metabolites with small number of hits in AntiBase

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Discussion

16 14 12 10 8 6

4 Number ofmetabolites Number 2

BG5

CTF9

CRF1 CRF2

SCF18 SCF25 SCF31

RSF17 RSF18 RSF23

CTF13 CTF14 CTF15 CTF20 CTF23 CTF24 CTF25

CRF11 CRF14 CRF17

Figure 9.5 Comparison of the isolates on the basis of the number of metabolites produced by each isolate as detected in HPLC-MS analysis, (red bars represent the isolates selected for preparative screening) made on the basis of gross biological and chemical screening results. The Streptomyces isolates RSF18, CRF17, BG5, CTF9, CTF15 and SCF25 were selected on the basis of their distinctive characteristics in pre screening studies, for fermentation, isolation, purification and structure elucidation of the active metabolites produced by them (Table 9.1; Fig 9.5). Moreover the established enormous diversity of metabolic products among our isolates, predict that many potentially new metabolites may be obtained in further research on this indigenous Streptomyces strain collection. Also the results indicate that the biological and chemical screening is a simple strategy for recognizing the unique activities, the specific metabolic fingerprint of an isolate, highlighting the particular metabolic characteristics of the isolate with respect to the other related strains or simply to recognize a competent strain from a strain collection.

For preparative screening it was necessary to cultivate the strains on larger scale, so the selected isolates were cultivated up to 25-50 liters, as shaking cultures or in lab fermenters. It was observed that the strains cultivated in flasks as shaking cultures exhibited more promising growth, high yields of crude extracts and structurally more stable metabolites as compared to the strains cultivated in lab fermenters. The culture broths were filtered by passing through a filter press and the mycellial cake and aqueous phase were extracted separately to get the crude extracts. The active components in a 218

Discussion crude extract were purified by silica gel column chromatography, preparative TLC and gel exclusion chromatography. The purified fractions were identified by mass spectrometry and NMR spectroscopy, along with comparisons of the data with different data bases like AntiBase (Laatsch AntiBase 2007) and dictionary of natural products (DNP) which is a most comprehensive database of natural products. Virtually all the famous structural classes of the compounds, including aminoglycosides, peptides, macrolides, quinones, polyethers etc were isolated from these Streptomyces isolates. It is worth mentioning here that the compounds detected in chemical screening were isolated from some of the isolates in preparative screening, however it was not true for all the screened isolates. For example the compounds with molecular masses m/z 1131 and m/z 1115 were detected in the chemical screening of the isolate RSF18. The isolated compounds from the culture broth of strain RSF18 are the, cyclic thiopeptides: geninthiocin (m/z 1132.3 M+H+) and the new val-geninthiocin (m/z 1116.33 M+H+) along with the macrolide chalcomycin (M/z 701.37 M+H+ not detected in chemical screening), it shows that this strain produced almost similar metabolites when it was grown as 1 liter culture (in prescreening) and as 15 liter shaking culture (in preparative screening). All the three purified compounds exhibited activity against Gram positive bacteria and minor antifungal activity, the same type of activities were exhibited by the crude extract of this strain in prescreening. The maximum yield of the compounds geninthiocin and the new val-geninthiocin produced by the strain RSF18 was obtained on the medium containing glucose as carbon source and yeast extract as nitrogen source o along with CaCl2 as a mineral salt, at pH-7.8, temperature 35 C and aeration at 110 rpm, in cultural optimization studies. The strain CRF17 in 16s rDNA sequencing showed 98% homology with Streptomyces pulcher, in prescreening it exhibited activities against both Gram positive and gram negative bacteria and strong antialgal activity, however the compound isolated from its culture broth by fermentation in a 20 L lab fermenter i.e. alborexin, is active against only Gram positive bacteria. The variation in activity might be due to the different growth behavior of the strain in a shaking flask and in a lab fermenter, and it is quite possible that the compounds responsible for the activity against gram negative bacteria and antialgal activity in prescreening could not be produced in fermenter or are being produced in low concentration that could not be isolated in

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Discussion preparative screening. The strain BG5 exhibited activities against both Gram positive and Gram negative bacteria along with antifungal activities in prescreening, however its preparative screening yielded ochromycenone, emycin D and 1-acetyl β-carbolin, which are active against Gram positive bacteria, the observed antifungal activities in prescreening of this strain could be attributed to β-carbolin analogues in low concentration which could not be isolated in preparative screening. In 16s rDNA sequencing the strain BG5 showed maximum similarity 98% with Streptomyces matensis. Margalith et al. (1959) first of all reported this specie of the genus Streptomyces, later this specie was also described by Shirling and Gottlieb (1968). The compounds ochromycenone, emycin D and 1-acetyl β-carbolin are reported to be produced by different species of the genus Streptomyces e.g ochromycenone has been isolated from the cultures of Streptomyces sp. GW71/2497 (Maskey et al., 2003), similarly emycin D has been isolated from the cultures of Streptomyces cellulosae (Rohr and Thiericke 1992; Gerlitz et al., 1995) and β-carbolins have been extensively isolated as plant metabolite and also from some Streptomyces strains (Shabaan et al., 2007). The strain CTF9 showed 99% similarity with Streptomyces malachitofuscus by 16s rDNA sequencing, it yielded phenyl acetic acid and β-indole lactic acid by fermentation in a 50 liter fermenter and by subsequent isolation and purification of the culture broth. In prescreening the strain CTF9 exhibited activity against almost all the tested organisms including bacteria, fungi and microalgae, it was expected that it might produce some interesting compounds, however in preparative screening only the phenyl acetic acid and β-indole lactic acid could be identified, the other purified compounds were found to be unstable and were difficult to identify, it might be possible that the broad spectrum activity of this isolate is due to the presence of these unidentified compounds in its culture broth, moreoever phenyl acetic acid and β-indole acetic acid also exhibit antibacterial and antifungal activities (Kim et al., 2004). In cultural optimization studies the maximum yield of the metabolites of strain CTF9 was obtained on the medium containing glucose as carbon source and yeast extract as nitrogen source along with CaCl2 as a mineral salt, at pH-6.5, temperature 35oC and aeration at 110 rpm. The strain CTF15 exhibited 99% similarity with Streptomyces grisioincarnatus by 16s r DNA sequencing, in prescreening this isolate exhibited activity only against Gram positive bacteria but high level of

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Discussion cytotoxicity almost 92% mortality of the shrimp larvae, the preparative screening results are in agreement with these activities and high cytotoxicity of the strain, since the well known compounds resistomycine, tetracenomycenone and actinomycin D were isolated from the culture broth of this strain, these compounds exhibit activities against Gram positive bacteria and are famous for their high cytotoxicity. The strain SCF25 in 16s rDNA sequencing exhibited 99% similarity with Streptomyces macrosporeus, in prescreening this strain exhibited activities against Gram positive and Gram negative bacteria, minor antifungal and antialgal activites, along with high cytotoxicity almost 91% mortality of the shrimp larvae. Again the expectation here was to find some unique or interesting compounds due to the broad spectrum activity of the strain, however three trivial compounds tryptophol, ferulic acid and tyrosol could be identified from its culture broth by fermentation in a 50 liter lab fermenter and by subsequent isolation and purification. The preparative screening results of all the investigated isolates support the idea of bioassay guided screening of a strain collection, as in most of the cases the activities exhibited by a strain in prescreening were confirmed by the metabolites purified from its culture broth.

Subjection of microorganisms to mutagenesis, followed by suitable selection and screening of the survivors has been a very effective tool in improving many industrial microorganisms. Variety of physical and chemical mutagens has been employed for enhancing the yield of the compounds of interest or for eliminating the unwanted characters of the producing strain. Shaw and Piwowarski (1977) suggested that ethidium bromide treatment eliminates the aerial mycelium and cause the loss of antibiotic production in Streptomyces. N-methyl-N'-nitro-N-nitrosoguanidine (MNNG or NTG) is considered as the most potent mutagen when used under appropriate conditions, (Kieser et al., 2003). Lee and Rho, 2002 reported that the yield of tylosin was enhanced up to 14 fold by treating the Streptomyces fradiae NRRL2702 by MNNG and short wavelength UV. Among the ionizing radiations gamma rays has been effectively used for obtaining mutants with significant increase in antibiotic production. Kenarova et al. 1996 reported that gamma irradiation caused three times increase in antimicrobial activity of Streptomyces hygroscopicus. The strain Streptomyces sp. RSF18 was selected for studying the effects of the selected chemical and physical mutagens on its growth and

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Discussion

20 18 16 14 12 10 NA 8 RA 6

Number mutants Number of SA 4 2 EA

1 2 5

10 15 10 20 50 30 45 60 20

0.5

250 100 500 750 -1 1000 μg ml μg ml-1 Minutes Gy EB MNNG UV Gamma Rays

Fig 9.6 Frequencies of the mutants of parental strain RSF18 exhibiting variable antimicrobial activity, obtained by different mutagenic treatments. NA= complete loss of activity (no activity), RA= reduced activity as compared to the parental strain, SA= same activity as compared to the parental strain, EA= enhanced activity as compared to the parental strain. antibiotic production ability. The motive for the selection of this strain for mutational analysis was its metabolic profile, it produces cyclic thiopeptides, the geninthiocin and val-geninthiocin along with the macrolide chalcomycin. Significant variations in the antimicrobial activity of the derivatives of the wild strain were observed in all the treatments with four different mutagenic agents. In treatments with different concentrations of ethidium bromide (10μg/ml, 20μg/ml, 50μg/ml, 100μg/ml for 1 hour), very small number of survivors could be recovered and in most of the cases reduction or loss in activity was observed, however some of the derivatives exhibited about 28% increase in activity, similarly some of the selected mutants e.g. the mutants 20.20EB/8, 29.20EB/10, 38.50EB/8, 43.50EB/10 exhibited up to 42% increase in activity against Bacillus subtilus. In treatments with low doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (250 μg/ml, 500 μg/ml, 750 μg/ml and 1000 μg/ml for 2 hours) most of the derivatives exhibited about 42% increase in activity however some of the mutants e.g. 70.500MNNG/10, 103.750MNNG/8 and 130.750MNNG/10 exhibited about 57% increase in the antimicrobial activity as compared to the parental strain against Bacillus

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Discussion subtilus. In treatments with UV at 254 nm (for 15 min, 30 min, 45 min, and 60 min) almost 42% increase in antimicrobial activity of different derivatives was observed. Similarly in treatments with different doses of gamma radiations (0.5 grays, 1.0 gray, 2.0 grays, 5.0 grays 10 grays and 20 grays), up to 28% increase in activity was observed in most of the cases, however some of the derivatives exhibited more than 42% increase in activity against Bacillus subtilus. The enhanced activity of the mutants selected in all the mutagenic treatments can be attributed to corresponding increase in the yield of the metabolites produced by the parental strain Streptomyces sp. RSF18. As a comparative account the treatments with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) at concentrations 500 μg ml-1 and 750 μg ml-1 yielded highest frequencies of the mutants with enhanced antimicrobial activity than all other mutagenic treatments (Fig 9.6). The treatments with MNNG not only yielded high number of chloramphenicol resistant mutants but also the maximum increase (up to 57%) in the activity of the wild strain Streptomyces sp. RSF18 as compared to the other mutagenic treatments. The results also depicts that the mutagenic agent like ethidium bromide, UV at 254 nm and gamma radiations have almost similar impact on the activity of the strain Streptomyces sp. RSF18. In all the cases it is still needed to find out the genetic variations in the selected mutants as compared to the parental strain, this aspect is still in progress in our ongoing studies on the characterization of the genes and biosynthesis pathways of the cyclic thiopeptides (the geninthiocin and val geninthiocin) produced by the strain Streptomyces sp. RSF18. On the whole the study reveals that the soil of this area Punjab, Pakistan is a promising source of inimitable bioactive streptomycetes, which should be continuously isolated, characterized and investigated for their antagonistic aptitude. The broad spectrum activity of the isolates against almost all classes of the pathogens including Gram positive bacteria, Gram negative bacteria, fungi and microalgae and the distinctive metabolic finger prints of the isolates in chemical screening, suggested them as a gorgeous source of competent microbial flora for antibiotics screening. Shortly it can be concluded that the indigenous streptomycetes are a potential source of interesting antimicrobial agents and their continuous exploration may yield novel metabolites which can be helpful to combat the uprising problem of antibiotic resistance among the pathogenic species.

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CHAPTER 10 REFERENCES

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Annexure

Publications and Presentations

This research work has been presented in many international conferences in different countries, Pakistan, Germany, Egypt and China. Two articles have been published in the journals of international repute and some are in preparation phase. Published Articles 1. Sajid, I., Shaaban, K, A., Frauendorf, H., Hasnain, S., Laatsch, H., 2008. Val- genenthiocin: Structure Elucidation and MSn Fragmentation of Thiopeptide Antibiotics Produced by Streptomyces sp.RSF 18, Z. Naturforsch, 63b, 1223- 1230. 2. Sajid, I., Yao, C. B., Shaaban,K, A., Hasnain, S., Laatsch, H., 2008. Antifungal and antibacterial activities of indigenous Streptomycetes isolated from saline farmlands, prescreening, ribotyping and metabolite diversity. World J. Microbiol. Biotechnol. 25(4): 601-610. (DOI 10.1007/s11274-008-9928) Conferences in which the work was presented 1. 1st GOTTINGER CHEMIE-FORUM, June 2007, Georg-August Universitat Gottingen, Germany. 2. GOTTINGER-TUBINGER, Natural Products Conference, Sep 2007, Eberhard- Karl State University Tubingen, Germany. 3. 1st Symposium of Microbiology and Molecular Genetics, on Genomics, proteomics, Metabolomics: Recent trends in Biotechnology, Oct 2007, Lahore, Pakistan. 4. “Funding research possibilities in developing countries” biannual international conference of TWAS/Bio Vision Alexandria, Egypt, April 11-16, 2008. 5. Second national conference on “Health Biotechnology” 27-28 May, 2008. Organized by “National Commission of Biotechnology” Islamabad, Pakistan. 6. 13th International Biotechnology Symposium and Exhibition, IBS 2008, (Biotechnology for the Sustainability of Human Society) 12-17 Oct 2008, Dalian China.

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Published Abstracts 13-O-008 1. Sajid, I., Shaaban, K, A., Frauendorf, H., Hasnain, S., Laatsch, H., 2008. Val- genenthiocin: A thiopeptides antibiotic produced by Streptomyces Sp. RSF18. Journal of Biotechnology, Abstracts of the Thirteenth International Biotecnology Symposium & Exhibition, 12-17 Oct, 2008, Dalian China. pp. S76 13-P-009 2. Sajid, I., Yao, C. B., Shaaban,K, A., Hasnain, S., Laatsch, H., 2008. Antifungal and antibacterial activities of indigenous Streptomycetes isolated from saline farmlands, prescreening, ribotyping and metabolite diversity. Journal of Biotechnology, Abstracts of the Thirteenth International Biotecnology Symposium & Exhibition, 12-17 Oct, 2008, Dalian China. pp. S79

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World J Microbiol Biotechnol (2009) 25:601–610 DOI 10.1007/s11274-008-9928-7

ORIGINAL PAPER

Antifungal and antibacterial activities of indigenous Streptomyces isolates from saline farmlands: prescreening, ribotyping and metabolic diversity

Imran Sajid Æ Clarisse Blandine Fotso Fondja Yao Æ Khaled Attia Shaaban Æ Shahida Hasnain Æ Hartmut Laatsch

Received: 26 July 2008 / Accepted: 19 November 2008 / Published online: 4 December 2008 Ó Springer Science+Business Media B.V. 2008

Abstract A culture collection of 110 indigenous Strepto- and new antifungal and antibacterial molecules are neces- myces strains originally isolated from saline farmlands sary to combat these pathogens (Fguira et al. 2005). Soil, in (Punjab, Pakistan) using stringent methods was screened particular, is an intensively exploited ecological habitat, the biologically and chemically to investigate their potential for microorganisms of which produce many useful natural the production of bioactive secondary metabolites. In a products, including clinically important antibiotics (Omura biological screening the crude extracts obtained from the 1992). Among soil inhabitants, actinomycetes, and more culture broth of selected strains were analysed for their specifically, streptomycetes, are of practical importance: activity against a set of test organisms, including Gram- these filamentous soil bacteria are rich sources of a large positive, Gram-negative bacteria, fungi and microalgae number of bioactive natural products with biological using the disk diffusion bioassay method. Additionally a activity, which are extensively used as pharmaceuticals and cytotoxicity test was performed by means of the brine shrimp agrochemicals. Streptomycetes produce about 75% of the microwell cytotoxicity assay. In a chemical screening each commercially and medically useful antibiotics and a major of the crude extracts was analysed by TLC using various fraction of the compounds developed for agricultural use staining reagents and by HPLC-MS/MS measurements. The has also been isolated from them (Lazzarini et al. 2001; results depicted an impressive chemical diversity of crude Takahashi and Omura 2003; Berdy 2005). extracts produced by these strains. The taxonomic status of In the screening for new bioactive microbial compounds, the selected strains was confirmed by preliminary physio- three major factors must be considered, i.e., selection of logical testing and 16S rRNA gene sequencing. producing organisms, culture methods and detection of metabolites. The screening process from isolation of the Keywords Indigenous Streptomyces spp Prescreening producing microbe to recognition of a new active metabolite, Ribotyping Metabolite diversity HPLC-MS/MS earlier took up to one year but has shrunken to a few days, using elevated methods (Laatsch 2000). The probability of success expected for the first stage is based on the singularity Introduction of the microorganism and the novelty of both specific activity and chemical structures. In order to elucidate out- The antibiotic resistance of a large number of pathogenic standing natural molecular diversity for drug development, bacteria and fungi is presently an urgent focus of research, various methods including target-directed biological, physico-chemical and chemical screening strategies have been developed (Broach and Thorner 1996; Grabley et al. 1999). I. Sajid S. Hasnain (&) Department of Microbiology and Molecular Genetics, University Chemical screening using TLC with various staining of the Punjab, Quaid-e-Azam Campus, Lahore 54590, Pakistan reagents and HPLC-MS/MS offers the opportunity to visu- e-mail: [email protected] alize a nearly complete picture of a microbial secondary metabolite pattern (metabolic finger-print) (Taddei et al. I. Sajid C. B. Fotso Fondja Yao K. A. Shaaban H. Laatsch Institute of Organic and Biomolecular Chemistry, University 2006; Burkhardt et al. 1996). This approach can be used of Go¨ttingen, Tammannstrasse 2, 37077 Gottingen, Germany advantageously for both, the detection of so-called 123 602 World J Microbiol Biotechnol (2009) 25:601–610

‘‘talented’’strains (Burkhardt et al. 1996), and for qualifying the selected strains for 7 days at the appropriate growth microbial strain collections, especially as a fundamental step temperature, an agar cylinder was cut out and shifted to LB of efﬁciently applied biological high-throughput assays. plates covered by 3 ml of top agar containing 50 llofa In this study we isolated bioactive Streptomyces strains 15 h culture of Bacillus subtilis and E. coli test strains, from soils of saline agricultural farmlands of different areas respectively, and then incubated overnight at 37°C. For of the Punjab, Pakistan. The selected strains were culti- antifungal activity, plates were covered by 3 ml of top agar vated on a small scale as one-liter shaking cultures and the containing 100 ll of spore suspension prepared from Mucor crude extracts of the bioactive compounds obtained with sp. The selected Streptomyces strains were stored as 5 ml ethyl acetate were screened biologically and chemically. In aliquots in the vapor phase of liquid nitrogen. a biological screening the extracts were analysed for their activity against a set of test organisms, including Gram- Cultivation of strains positive bacteria, Gram-negative bacteria, fungi and microalgae using the disk diffusion bioassay method. The selected strains were ﬁrst grown as subcultures on agar

Additionally a cytotoxicity test was performed using brine plates for 3 days using M2 agar. The resulting sub-cultures shrimp microwell cytotoxicity assay. In a chemical were used to inoculate 4 9 250 ml of M2 broth in 1-l screening each of the crude extracts was analysed by TLC Erlenmeyer flasks (the pH was adjusted to 7.8 before and HPLC in combination with tandem mass spectrometry sterilization) and the flasks were incubated as shaker cul- (MS/MS), providing an impression of the chemical diver- ture (95 rev/min) for 7 days at 28°C. After incubation the sity produced by these strains. From soil samples collected culture broth was lyophilized (Christ Alpha 2–4 LD) and at different sites in Punjab, 110 bioactive isolates were the resulting residue was mixed with ethyl acetate and was obtained. From this collection, twenty representative sonicated (Ultraturrax: Janke & Munkel KG) for 15 min. strains were selected for elucidating the metabolic diversity Later the mixture was filtered over celite bed, and the fil- and were identified as species belonging to the genus trate obtained was evaporated on a rotary evaporator to Streptomyces by analyzing their morphological character- obtain the crude extracts. The extracts were subjected to a istics (Locci 1989) and by preliminary physiological combination of chemical, biological, pharmacological testing (Wendisch and Kutzner 1991), 16S rRNA gene screening and HPLC-MS analysis. sequencing provided the final taxonomic status of these strains. Biological screening

Antimicrobial activity test Materials and methods The antimicrobial activity was determined by disk diffusion Selection of bioactive Streptomyces strains bioassays against a set of test organisms including Gram- positive bacteria (Staphylococcus aureus, Bacillus subtilis, Almost 30 soil samples were collected from salt-affected Streptomyces viridochromogenes Tu¨57), Gram-negative areas of the Punjab in sterile bags, the soil samples were bacteria (E. coli), fungi (Candida albicans and Mucor treated following the physical and chemical treatment miehei) and microalgae (Chlorella sorokiniana, Chlorella methods of Hayakawa et al. 2004. One gram of each soil vulgaris and Scenedesmus subspicatus). The test plates for sample was suspended in 10 ml of sterile water and vor- bacteria were prepared by pouring 14 ml of L-agar (Gerhardt texed for 45 s. The samples were serially diluted and 50 ll et al. 1994) as base layer; after solidifying, this was overlaid of the dilution 10-3 was spread over the surface of Casein– with 4 ml of the inoculated seed layer. The test plates for

KNO3 agar (glycerol 10 g, KNO3 2 g, casein 0.3 g, NaCl fungi and Streptomyces viridochromogenes (Tu¨57) were 2g,K2HPO4 2 g, MgSO4 7H2O 0.05 g, CaCO3 0.02 g, prepared by pouring 14 ml of M2 medium as base layer. FeSO4 7H2O 0.01 g, agar 18 g in one liter, cycloheximide After solidifying, this was overlaid with 4 ml of the agar 50 lg/ml, as an antifungal agent). After incubation at 28°C inoculated with fungal spores as seed layer. Bold and basal for 7–21 days, Streptomyces colonies were selected and medium (Kantz and Bold 1969) were used for making algal transferred to fresh M2 agar (10 g malt extract, 5 g yeast test plates with the same procedure. Paper disks with diam- extract, 5 g glucose, 15 g agar in one liter of tap water). The eter of 9 mm were impregnated with 40 ll of crude extract

Streptomyces cultures were puriﬁed by repeated sub cul- solution (crude extracts 1 lg/ll dissolved in CH2Cl2/MeOH turing on M2 medium. A preliminary antimicrobial activity 1:1); the disks were dried under sterile conditions and placed test was performed using solid media bioassay method on the surface of test plates. The bacterial and fungal test against a standard of Gram-positive and Gram-negative test plates were incubated at 37°C for 16–24 h, while the algal bacteria and fungi. In a solid media test, after incubation of test plates were incubated at room temperature in artiﬁcial 123 World J Microbiol Biotechnol (2009) 25:601–610 603 daylight for 96 h. After incubation, the diameter of inhibition N = Number of the dead larvae before starting the test. zones was measured in mm. G = Total number of larvae.

Bioautography Identiﬁcation of the selected strains

The crude extract samples were fractionated on TLC plates The selected Streptomyces strains were characterized by eluting the plates with 5% CH2Cl2/MeOH solvent sys- morphologically and physiologically following the direc- tem. For each of the sample, the developed TLC plate was tions given by International Streptomyces Project (ISP) and cut into two parts with clean scissors, one of the half acted 16S rRNA gene sequencing. The physiological character- as reference TLC and was sprayed with spraying reagent ization includes the formation of melanin pigment and

(anisaldehyde/H2SO4), while the second half was fixed utilization of nine different sugars as carbon source. inversely over the surface of the test plate seeded with indicator microorganism. The test plate was incubated at 16S rRNA gene sequencing 37°C for 24 h. Inhibition zones were compared with the related spots on the reference TLC plate and the active High-molecular weight chromosomal DNA of the 10 fractions in a crude extract were marked. selected strains was prepared from M2 grown mycelia following the methods of Felnagle et al. (2007). PCR Brine shrimp microwell cytotoxicity assay amplification of the 16S rRNA gene of strains was done using two primers P1: 50-AGAGTTTGATCATGGC-30 and Cytotoxicity of the crude extracts was determined against P2: 50-TACCTTGTTACGACTT-30, as described by brine shrimps (Artemia salina) using a microwell cyto- Edwards et al. (1989). Approximately 300 ng genomic toxicity assay (Solis et al. 1993). The mortality rate was template DNA was used with 150 pmol of each primer per determined by the following procedure: 50 ll of reaction volume. To improve the denaturation of Dried eggs of Artemia salina (0.5 g) were added to a the DNA, 5% (v/v) DMSO was added to the reaction 500 ml separating funnel, filled with 400 ml of artificial mixture. Amplification was performed in an automated seawater. The suspension was aerated by bubbling air into thermocycler (Eppendorf Mastercycler) using 1U Pfu DNA the funnel and kept for 24–48 h at room temperature. After polymerase (Fermentas) and the recommended buffer aeration had been removed, the suspension was kept for 1 h system according to the following amplification profile: undisturbed, whereby the remaining eggs settled down. In 1, 94°C 5 min; 2, 94°C 1 min; 3, 55°C 1 min; 4, 72°C 2 min; order to use only the active larvae, one side of the sepa- 5, 72°C 5 min; 30 cycles. The PCR product was analysed by rating funnel was covered with aluminum foil and the other agarose gel electrophoresis and the DNA of the expected illuminated with a lamp, whereby the phototropic larvae size was purified, the sequencing was done using dye-ter- were gathering at the illuminated side. With a pipette, 30– minator chemistry with Mega-BACE 1,000/4,000 DNA 40 shrimp larvae were collected and transferred to a deep- automated sequencers (Amersham Bioscience). The 16S well microtiter plate (wells diameter 1.8 cm, depth 2 cm) rRNA gene sequence data obtained was first analysed filled with 0.2 ml of salt water. The dead larvae were using the advanced BLAST search program at the NCBI counted (value N), a solution of 20 lg of the crude extract website: http://www.ncbi.nlm.nih.gov/BLAST/. The nucle- in 5–10 ll of DMSO was added and the plate kept at room otide sequence data was deposited to the GenBank, and temperature in the dark. After 24 h, the number A of the GenBank accession numbers for 17 strains were obtained. dead animals in each well was counted again under the microscope. The surviving larvae were killed by addition Chemical screening of 0.5 ml methanol so that subsequently the total number G of the animals could be determined. Each test row was In the chemical screening, TLC in combination with accompanied by a blind sample containing pure DMSO spraying reagents and HPLC-MS methods were used. instead of a test solution. As a positive control with 100% mortality, actinomycin D (10 lg/ml) was used. The mor- Thin layer chromatography (TLC) tality rate M was calculated using the following formula: ðÞA B N The crude extracts obtained from the selected strains were M ¼ 100 analysed by TLC. In this method a small drop of a sample ðÞG N was spotted onto the TLC plate with a capillary and dried; Where M = percent of the dead larvae after 24 h. the spotting process was repeated by superimposing more A = Number of the dead larvae after 24 h. B = Average drops on the original spot for obtaining appropriate quantity number of the dead larvae in the blind samples after 24 h. (2–5 lg) of the sample on the plate. The TLC plates were 123 604 World J Microbiol Biotechnol (2009) 25:601–610

developed with a CH2Cl2/5% MeOH solvent system. Zones The capillary temperature was adjusted at 220°C. The on the developed plates were visualized under u.v. light (at source was operated in both ion modes using nitrogen at 80 254 nm and 366 nm) and the components showing u.v. psi as sheath gas. The samples were scanned from 100 to absorption or fluorescence were marked. Later the TLC 2,000 amu. plates were sprayed with anisaldehyde/sulfuric acid or Mass spectrometer: Finnigan LCQ; u.v./VIS-Detector: Ehrlich’s reagent (detection of indoles) for the further Finnigan Surveyor PDA Detector (Thermo Electron Cor- localization of interesting zones. The colored bands (met- poration); HPLC pumps: Rheos 4000; Degasser: ERC- abolic finger-print) produced by the reaction between spray 3415a (both Flux Instruments); Autosampler: Jasco 851- reagent and the metabolites were marked and documented AS Intelligent Sampler (Jasco); HPLC control software: by scanning. Janeiro (Flux Instruments); File system: Xcalibur (Finni- gan); Database software: MS-Manager (ACDLabs); HPLC-MS column: EC 125/2 Nucleosil 100-5 C18 (Macherey–Na- gel), Synergi 4l MAX-RP 80A, 150 9 2.00 mm, 4 lm Sample preparation is a topic of high importance when an (Phenomenex); solvent: Methanol LiChrosolv hypergrade LC/MS/MS method is applied to assay biological samples. It for liquid chromatography (Merck) and water, both con- must ensure certain basic elements of ruggedness and containing 0.05% formic acid (to increase the sharpness and sistency that are expected in any assay. Generally a quality of the signals) was used. Program: start 10% concentration from 1 to 5,000 ng/ml is needed. The solvent methanol to 100% in 20 min, and maintained for 10 min at used was methanol/water. Methanol (LiChrosolv hypergrade 100% methanol. For the next run, the gradient was set for liquid chromatography) was purchased from Merck. down subsequently during 2 min to 10% methanol again and held there for 8 min for equilibration, flow rate: Chromatographic and mass spectrometric conditions 300 ll/min, injection volume 5 ll, detection range 200– 800 nm. For the chromatographic separation, an RP-C12 column (Synergi 4l MAX-RP 80A, 150 9 2.00 mm, 4 lm) from Phenomenex was used. RP-C12 possesses a lower number Results (25%) of free silanol groups and results in better separation characteristics, especially for basic and tailing compounds. In all 110 active Streptomyces strains were selected on the Electrospray Ionization (ESI) was applied in positive and basis of preliminary antimicrobial activity tests. From this in negative polarity at the electrospray voltage of 4.50 kV. collection, 20 strains were selected for detailed analysis

Fig. 1 Antimicrobial activities of selected Streptomyces extracts CTF9 (4) against E. coli. e Activity of RSF18 (5), SCF18 (6) and against different test organisms. a Activity of RSF23 (1), BG5 (2), CRF17 (7) against Bacillus subtilis. f Activity of SCF31 (1), CRF11 SCF25 (3) and CTF9 (4) against Candida albicans. b Activity of (2), CRF2 (3) and CTF20 (4) against Streptomyces viridochromog- RSF17 (1), RSF18 (2), SCF 18 (3) and CRF17 (4) against Mucor enes (Tu¨57). g Activity of RSF18 (1), SCF 18 (2), CRF17 (3) and miehei. c Activity of SCF31 (1), CRF11 (2), CRF2 (3) and CTF20 (4) RSF17 (4) against Chlorella sorokiniana. h Bioautography of strain against E. coli. d Activity of RSF23 (1), BG5 (2), SCF25 (3) and CRF17, test organism: Staphylococcus aureus 123 ol irbo itcnl(09 25:601–610 (2009) Biotechnol Microbiol J World

Table 1 Results of biological screening (antimicrobial activity and cytotoxicity tests) Streptomyces Antimicrobial activity test organisms zone of inhibition (mm) Cytotoxicity test organism strains (% age mortality) S. aureus B. subtilis S. viridochromogenes E. coli C. albicans M. miehei C. vulgaris C. sorokiniana S. subspicatus Artemia salina (Tu¨ 57)

RSF 17 14 16 13 14 ? 12 -? - 15.2 RSF 18 16 17 12 15 ---- - 13.5 RSF 23 11 - 16 - 16 ? 12 11 - 92.7 BG 5 16 20 27 19 27 11 12 12 - 28.9 CRF 1 12 11 13 ------23.5 CRF 2 13 11 ?------93.7 CRF 11 22 18 17 17 -?-- - 97.2 CRF 14 18 16 18 14 -?-- - - CRF 17 23 15 11 15 ? 18 -- 25 69.2 SCF 18 -- ? -? 17 -- - 78.4 SCF 25 25 22 20 20 20 ? 11 12 28 91.4 SCF 31 12 - 19 ?- - - - - 16.5 CTF 9 13 12 20 12 20 ? 16 18 12 11.5 CTF 13 11 ? 12 11 ---- - 75.4 CTF 14 15 11 11 ------100 CTF 15 19 13 11 ------91.2 CTF 20 12 13 17 12 -?-- - 65.0 CTF 23 ?? 11 ------CTF 24 ? 11 12 ------CTF 25 11 - 15 -? - - - - 0.7 (1) 5 Minor zone of inhibition around the disc, (-) = No activity against the test organism was observed, cytotoxicity not determined 123 605 606 World J Microbiol Biotechnol (2009) 25:601–610 depending on their origin, culture behavior and level of utilize L-arabinose as the carbon source; however, in case activity. The strains were found to be active against a of rafﬁnose six different strains showed growth on it (see variety of indicator microorganisms in biological screening Table 2). Comparison of the cultural characteristics of (Fig. 1). As seen in Table 1, the strains RSF23, BG5, these strains with those of known actinomycete species CRF17, SCF18, SCF25 and CTF9 exhibited antifungal described in Bergey’s Manual of Systematic Bacteriology activity against Candida albicans and Mucor miehei. (Lechevalier et al. 1989), strongly suggested that these Activity against microalgae (Chlorella sorokiniana, Chlo- strains belong to the genus Streptomyces. The BLAST rella vulgaris and Scenedesmus subspicatus) was exhibited analysis of 16S rRNA gene sequence data of the selected by the isolates CRF17, SCF25, CTF9 (Table 1, Fig. 1). strains showed alignments of these sequences with reported Almost all the strains were found to be active against Gram- 16S rRNA gene sequences in GenBank. The number of positive test bacteria (Staphylococcus aureus, Bacillus nucleotides sequenced for each strain and the highest subtilis and Streptomyces viridochromogenes-Tu¨57), how- similarities found with different species of the genus ever, activity against Gram-negative bacteria (E. coli) was Streptomyces and GenBank accession numbers for 17 exhibited only by ﬁve of the isolates i.e., RSF18, BG5, strains are summarized in Table 3. CRF11, CRF17, and SCF25. The minimum cytotoxicity In chemical screening, u.v.-absorbing spots were against Artemia salina was observed in case of isolates observed on TLC plates (Fig. 2a, b) by visualizing under CTF25 and RSF18, while the isolates SCF25, CRF2, and u.v. light at short and long wavelengths (254 nm, 366 nm). RSF23 were found to be highly cytotoxic (Table 1). The pattern of colored bands after treatment of TLC plates

In physiological testing, the strains showed melanin with staining reagents (anisaldehyde/H2SO4 and Ehrlich’s production and exhibited the ability to grow on different reagent) is visible in Fig. 2c, d. The constituents of each of carbon sources. Three out of 20 strains including CRF14, the crude extracts produced different colors including dark CTF23, and CTF24 did not produce melanin. All the brown, blue, pink and yellow depending on the staining strains were able to grow on glucose, fructose and man- reagent. The highest variety of colored bands was observed nose. Only two strains BG5 and CTF20 grew on in crude extracts of strains SCF25, CRF11, CRF14, CTF9, D-galactose, while three strains SCF31, CTF9, CTF13 CTF13, CTF14, and CTF15. exhibited growth on sucrose. The strains CRF11, CRF14, In HPLC/MS analysis, each of the crude extracts exhib- CTF13, CTF14, CTF23, and CTF24 were found unable to ited a varying number of peaks at different retention times.

Table 2 Melanin production and sugar utilization proﬁle of the selected strains

Isolates Production of melanin D-Glucose D-Fructose L-Arabinose D-Galactose Rafﬁnose D-Mannitol Sucrose Mannose

RSF 17 ????-??-? RSF 18 ????-??-? RSF 23 ????-??-? BG 5 ?????-?-? CRF 1 ????--?-? CRF 2 ??11 2 ?? -? CRF 11 ??12 2 ?? -? CRF 14 -?12 2 -- -? CRF 17 ??11 2 ?? -? SCF 18 ??11 2 -? -? SCF 25 1 ?? ? - -? -? SCF 31 1 ?? ? - -? ?? CTF 9 ????--??? CTF 13 ???---??? CTF 14 ???---?-? CTF 15 ????--?-? CTF 20 ?????-?-? CTF 23 -??-----? CTF 24 -??-----? CTF 25 ????--?-? (?) = Produces melanin and utilizes the respective sugar as carbon source, (-) = No melanin production and no growth or limited growth

123 World J Microbiol Biotechnol (2009) 25:601–610 607

Table 3 GenBank accession Isolate No. of Nucleotides % homology with GenBank Accession numbers along with the sequenced (bp) Number alignments of sequences obtained with reported 16S RSF 17 1421 Streptomyces sp. 99 EU301834 rRNA gene sequences in GenBank and highest similarity RSF 18 1418 Stretomyces sp. 99 EU294139 with different Streptomyces RSF 23 976 S. chromofuscus 94 EU301837 species (17 strains) BG 5 1433 S. matansis 98 EU301836 CRF 1 721 S. macrosporeus 92 EU301829 CRF 2 1437 S. vinaceus 99 EU294133 CRF 11 859 S. heliomycini 97 EU301828 CRF 17 1420 S. pulcher 98 EU294134 SCF 18 1417 S. griseoincarnatus 98 EU294140 SCF 25 1425 S. macrosporeus 99 EU301835 SCF 31 867 S. malachitofuscus 85 EU294141 CTF 9 1426 S. malachitofuscus 99 EU294138 CTF 13 1390 S. erythrogriseus 99 EU301830 CTF 14 1393 S. labedae 99 EU294135 CTF 15 1429 S. griseoincarnatus 99 EU301831 CTF 20 1427 S. rochei 99 EU294136 CTF 25 1425 S. macrosporeus 98 EU301833

A For MS/MS measurements, a mass range of m/z =±1 amu was used. In most cases quasimolecular ions were found for [M ? H]?,[M? Na]?, [M–H]-, in addition to ions of dimers [2 M ? H]?,[2M? Na]? and [2 M–H]-, [2 M ? Na–2H]- etc. Sometimes the signal intensity of the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 dimer ion was higher than that of the monomer ion. To avoid B this, an optimization was done relating to the ion source collision-induced dissociation (ISCID), which allows the dimer to decay back into the monomer. At the end collision energy around 10 V was favored because of the stability of different molecules. Figure 3 depicts the HPLC-MS results 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 of the crude extract of Streptomyces sp. RSF18. The total ion C chromatogram (first graph) and the u.v. line (second graph from top) showed two main peaks at Rt 18.32 and 17.12 min with molecular masses of 1,115 and 1,131 Da, respectively. The selected ion chromatogram and a subsequent MS/MS measurement gave hints for a peptide with dehydroamino 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 acids (Fig. 4). The results of a search in AntiBase (Laatsch, D AntiBase 2007) using the masses, resulted in five hits for m/z = 1,131, one of them being the known thiopeptide antibiotic geninthiocin (Yun et al. 1994; see Fig. 5). The identity with the latter was confirmed by subsequent isolation and NMR spectroscopic characterization. For the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 remaining HPLC-MS data, between zero (in extract CRF11) and more than 100 hits (e.g., SCF18) were found for each Fig. 2 Chemical screening using TLC detection. TLC plates a under u.v. at 254 nm, b under u.v. at 366 nm, c after treatment with signal. The minimum number of hits in AntiBase was found anisaldehyde/H2SO4 sol., d after treatment with Ehrlich’s reagent. for the compounds of strains RSF17, RSF18, BG5, CRF11, Numbers 1–20: Crude extracts of Streptomyces strains 1 = RSF17, 2 = CRF17, and SCF31. The strains SCF18, CTF9, CTF15 were RSF18, 3 = RSF23, 4 = SCF18, 5 = SCF25, 6 = SCF31, 7 = CRF1, 8 = found to produce large numbers (more than 10) of metabo- CRF2, 9 = CRF11, 10 = CRF14, 11 = CRF17, 12 = BG5, 13 = CTF9, 14 = CTF13, 15 = CTF14, 16 = CTF15, 17 = CTF20, 18 = CTF23, 19 = lites. While a missing database entry indicates that the CTF24, 20 = CTF25 respective compound most likely has not been isolated from 123 608 World J Microbiol Biotechnol (2009) 25:601–610

Fig. 3 Total ion chromatogram RT: 0,00 - 30,06 24,89 of crude extract RSF18 100 NL: 5,12E7 90 Base Peak 80 MS RSF18dp1 70 9,28 60 9,58 50 16,00 20,06 17,12 40 24,81 8,41 9,70 18,32 25,05 7,18 11,24 15,78

Relative Abundance 30 26,03 20 1,05 6,99 21,59 3,77 10 11,75 15,50 29,15 0 1,35

NL: 3,45E5 300000 17,05 Total Scan PDA 250000 RSF18dp1 18,24 200000 10,03 uAU 150000 5,65 1,79 6,51 16,23 100000 7,98 13,75 3,55 10,96

50000 19,98 20,24 22,43 24,83 27,84 0 0 5 10 15 20 25 30 Time (min) micro-organisms before, a higher number is not more than a suggested them as a good source of interesting antibacterial, measure for the probability to re-isolate known metabolites. antifungal and anti-microalgal compounds. As a comparative account the strains CRF17, SCF25, CTF9, and BG5, later identified as Streptomyces pulcher, Streptomy- Discussion ces macrosporeus, Streptomyces malachitofuscus, and Streptomyces matensis, respectively, were found to deliver The present work describes antimicrobial activities and highly active extracts, which showed a broad spectrum of metabolic diversity of the Streptomyces flora of saline agri- activities (Table 1, Fig. 1). It has been estimated that the cultural farmlands. The cultural and morphological genus Streptomyces might produce at least 100,000 new characteristics of the selected strains strongly suggested that compounds of biological interest (Watve et al. 2001). they belong to the genus Streptomyces. Later the active During screening of new isolates for drug discovery, many strains were identified by preliminary physiological testing potentially interesting microorganisms might be excluded and 16S rRNA gene sequencing. In physiological testing the due to inadequate techniques, too high selectivities or just strains were conspicuous for pigment production and their because of missing tests (Taddei et al. 2006). We decided ability to utilize different carbon sources (Table 2). The 16S therefore, to use a so-called horizontal screening, i.e., a rRNA gene sequencing proved that all the isolates were combination of tests with low selectivity covering a broad different species of the genus Streptomyces. The GenBank range of antibacterial, antifungal, phytotoxic and cytotoxic accession numbers of these Streptomyces species are given activities (Laatsch 2000). Additionally we utilized the in Table 3. It seems important to mention here that our major chemical screening approach to establish similarities or interest was to investigate the potential of our collection of differences between the secondary metabolite patterns of Streptomyces strains (110 strains) for the production of our isolates assuming that each strain produces a specific unique or rare bioactive compounds, so 20 representative metabolic fingerprint when it is grown under the same strains were selected depending on their different origin and culture conditions. Chemical screening by TLC using were subjected to biological and chemical screening. selected spraying reagents and HPLC-MS/MS analysis Biological screening results proved the strains as potent showed a pattern of secondary metabolites specific for each producers of bioactive secondary metabolites and clearly strain and provided deep insight into the metabolites of a 123 World J Microbiol Biotechnol (2009) 25:601–610 609

Fig. 4 Selected ion chromatogram of extract RSF18 (ﬁrst line), u.v. chromatogram, mass spectrum of the signal of geninthiocin at Rt = 17.12 min, and MS/MS fragmentation (bottom) of the ion signal [M ? Na] at m/z 1154

O O H stain the various spots with different colors. The charac- N teristically colored bands produced on TLC plates were N NH2 N H marked and documented by scanning (Fig. 2). HPLC-MS O S analysis of the crude extracts produced by these strains gave an additional picture of their metabolic diversity. The O N N molecular masses resulting from HPLC-MS measurements O of the crude extracts were searched in AntiBase, which is a O NH database for rapid structure determination of microbial natural products (Laatsch, AntiBase 2007): A hit number[0 NH HN O in AntiBase reflects a high probability that the compound HO may already be known; in contrast, a hit number of 0 NH O N (occurred only once, for Rt 18.53 in CRF11) indicates O clearly that the compound in question has probably not been H isolated from micro-organisms before. HO N O N O As HPLC-MS alone did not provide satisfactory infor- H N O mation for a complete dereplication (= rapid identification N H of known compounds), we applied additionally tandem O mass spectrometry (MS/MS), i.e., the quasimolecular ions Fig. 5 Molecular structure of geninthiocin isolated from the Strep- were bombarded with helium atoms at a collision energy of tomyces Sp. RSF18 35% of the instrument’s maximum. The fragmentation pattern especially of peptides is highly structure-sensitive strain ensuring its qualification or disqualification for fur- and allows to determine the amino acid sequence often ther work. In case of thin layer chromatography (TLC, quite easily. For the rapid identification of trivial com-

CH2Cl2/5% MeOH as solvent system), anisaldehyde/H2SO4 pounds, the resulting fragment pattern was compared with as spray reagent gave the best results, due to its capability to our MS/MS-database of about 600 frequently occurring 123 610 World J Microbiol Biotechnol (2009) 25:601–610 microbial metabolites. In this way, e.g., diketopiperazines, Edwards U, Rogall T, Bloecker H, Emde M, Boettger EC (1989) constituents of the nutrient broth (e.g., daidzein, genistein) Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. or contaminants (e.g., phthalic esters) can be easily iden- Nucleic Acids Res 17:7843–7853. doi:10.1093/nar/17.19.7843 tified. In extracts of strain CTF15, cis-cyclo(prolylvalyl) Felnagle EA, Rondon MR, Berti AD, Crosby HA, Thomas MG and cis-cyclo(prolylleucyl) were easily identified; other (2007) Identification of the biosynthetic gene cluster and an compounds with similarity index between 50 and 70% additional gene for resistance to the antituberculosis drug capreomycin. Appl Environ Microbiol 73:4162–4170. doi: were tetracenomycin D, isoxanthohumol, resistoflavine, 10.1128/AEM.00485-07 versicolorin C, resistomycin, lagumycin A, and diazaqui- Fguira LF, Fotso S, Mehdi RA, Mellouli L, Laatsch H (2005) Purification nomycin. Indeed, the preparative separation delivered and structure elucidation of antifungal and antibacterial activities of resistomycin and tetracenomycin D. a newly isolated Streptomyces sp strain US80. Res Microbiol 156(3):341–347. doi:10.1016/j.resmic.2004.10.006 Further dereplication is possible by comparison of u.v. Gerhardt P, Murry RGE, Wood WA, Kreig NR (1994) Methods for absorptions or the empirical formula (obtainable by general and molecular bacteriology. ASM Washington, DC HRMS) of the respective signal with data collections (e.g., Grabley S, Thiericke R, Zeeck A (1999) The chemical screening AntiBase), or by selective chemical reactions: If developed approach. In: Grabley S, Thiericke R (eds) Drug discovery from nature. Springer–Verlag, Heidelberg, pp 124–148 chromatograms of the crude extracts are moistened with Hayakawa M, Yoshida Y, Iimura Y (2004) Selective isolation of 2 M NaOH, a color change of yellow or orange spots to bioactive soil actinomycetes belonging to the Streptomyces - blue or violet indicates the presence of peri-hydroxyqui- violaceusniger phenotypic cluster. J Appl Microbiol 96:973– nones, as it was the case for strains SCF25, CRF11, 981. doi:10.1111/j.1365-2672.2004.02230.x Kantz T, Bold HC (1969) Morphological and taxonomic investigation CRF14, and BG5. Other chemical reactions are indicative of Nostoc Anabena in culture. Physiological studies, University for peptides, prodigiosins, alkaloids, etc. of Texas (publication No. 6924), Austin, Texas, USA Using this approach of chemical screening including Laatsch H (2000) Mikroorganismen als biologische Quelle neuer TLC, HPLC-MS/MS and database searches, we were able Wirkstoffe. In: Kayser O, Mu¨ller RH (eds) Pharmazeutische Biotechnologie. Wissenschaftliche Verlagsges, Stuttgart, to establish the enormous diversity of metabolites of the Germany, pp 13–43 isolates. As a consequence of the detailed analysis of the Laatsch H (2007) AntiBase 2007, a data base for rapid dereplication results obtained from our biological and chemical screening and structure determination of microbial natural products. project it seems reasonable to comment on the secondary Wiley–VCH: Weinheim, Germany (and annual updates); see http://wwwuser.gwdgde/*ucoc/laatsch/AntiBase.htm metabolism of the analysed strains and on our whole Lazzarini A, Cavaletti L, Toppo G, Marinelli F (2001) Rare genera of Streptomyces collection (110 strains) in a more general actinomycetes as potential sources of new antibiotics. Antonie manner. Regarding their metabolic fingerprint visualized by Van Leeuwenhoek 78:399–405. doi:10.1023/A:1010287600557 the chemical screening it is possible to select the talented Lechevalier HA, Williams ST, Sharpe ME, Holt JG (1989) The Actinomycetes: a practical guide to genetic identification of organisms from this collection, which may produce rare or a actinomycetes. In: Bergey’s manual of systematic bacteriology. large variety of structurally diverse secondary metabolites. pp 2344–3330 Additionally the spectrum of antimicrobial activity Locci R (1989) Streptomycetes and related genera. In: Williams ST, observed in biological screening depicts that these Strep- Sharpe ME, Holt JG (eds) Bergey’s manual of systematic bacteriology. Williams & Wilkins, Baltimore, pp 2451–2493 tomyces strains could act as a potential source of the Omura S (1992) The search for bioactive compounds from micro- compounds with broad spectrum activity, for controlling organisms, 1st edn. Springer, Berlin, Heidelberg, New York bacterial, fungal, and algal pathogens. Solis PN, Wright CW, Anderson MM, Gupta MP, Phillipson JD (1993) A microwell cytotoxicity assay using Artemia salina (brine Acknowledgments We are thankful to Dr. H. Frauendorf for mass shrimp). Planta Med 59:250–252. doi:10.1055/s-2006-959661 spectra, and F. Lissy and A. Kohl for technical assistance. A financial Taddei A, Valderrama M, Giarrizzo J, Rey M, Castelli C (2006) support of this work by a grant from Higher Education Commission Chemical screening: a simple approach to visualizing Strepto- (HEC) of Pakistan under IRSIP is gratefully acknowledged. myces diversity for drug discovery and further research. Res Microbiol 157:291–297. doi:10.1016/j.resmic.2005.07.007 Takahashi Y, Omura S (2003) Isolation of new actinomycete strains for the screening of new bioactive compounds. J Gen Appl Microbiol 49:141–154. doi:10.2323/jgam.49.141 References Watve MG, Tickoo R, Jog MM, Bhole BD (2001) How many antibiotics are produced by the genus Streptomyces? Arch Microbiol 157:386– Berdy J (2005) Bioactive microbial metabolites: a personal view. J 390. doi:10.1007/s002030100345 Antibiot 58(1):1–26 Wendisch FK, Kutzner HJ (1991) The family Streptomycetaceae. In: Broach JR, Thorner J (1996) High throughput screening for drug Balows A, Tru¨per HG, Dworkin M, Harder W, Schleifer KH discovery. Nature 384:14–16. doi:10.1038/384014a0 (eds), The prokaryotes, A handbook on the biology of bacteria: Burkhardt K, Fiedler HP, Grabley S, Thiericke R, Zeeck A (1996) New ecophysiology, isolation, identification, applications. Springer– cineromycins and muscacins obtained by metabolic pattern analysis Verlag, Berlin/New York, pp 965–968 of Streptomyces griseoviridis (FH-S 1832) I. Taxonomy, fermen- Yun BS, Hidaka T, Furihata K, Seto H (1994) Microbial metabolites tation, isolation, and biological activity. J Antibiot 49:432–437 with tip A promoter inducing activity. J Antibiot 47:969–975

123 Val-Geninthiocin: Structure Elucidation and MSn Fragmentation of Thiopeptide Antibiotics Produced by Streptomyces sp. RSF18

Imran Sajida,b, Khaled A. Shaabana, Holm Frauendorfa, Shahida Hasnainb, and Hartmut Laatscha a Department of Organic and Biomolecular Chemistry, University of G¨ottingen, Tammannstraße 2, D-37077 G¨ottingen, Germany b Department of Microbiology and Molecular Genetics, University of Punjab, Quaid-i-Azam Campus, Lahore 54590, Pakistan Reprint requests to Prof. Dr. H. Laatsch. Fax: +49(0)551-399660. E-mail: [email protected]

Z. Naturforsch. 2008, 63b, 1223 – 1230; received May 23, 2008

Val-Geninthiocin (2), a new member of thiopeptide antibiotics, was isolated from the mycelium of Streptomyces sp. RSF18, along with the closely related geninthiocin (1) and the macrolide, chalcomycin. By intensive NMR and MS studies, Val-geninthiocin (2) was identiﬁed as desoxy- geninthiocin, a thiopeptide, containing several oxazole and thiazole units and a number of unusual amino acids. Compound 2 shows potent activity against Gram-positive bacteria and minor antifungal activity, while it is not effective against Gram-negative bacteria or microalgae. Here we describe the fermentation, isolation and structure elucidation as well as the biological activity of 2.

Key words: Val-Geninthiocin, Thiopeptide Antibiotic, Streptomyces

Introduction Thiopeptide antibiotics are forming a group of cyclic peptides characterized by several common structural features, such as oxazole and thiazole units and unusual amino acids; especially dehydroamino acids are typical [1, 2]. Thioxamycin [3], berninamycin A [4], sulfomycin I [5 – 7], and A10255 [8] e. g. possess the thiazole-pyridine-oxazole substructure, while thiocillin I [9, 10], micrococcin P [11] and GE2270 A [12] are characterized by a thiazole- pyridine-thiazole moiety. Thiopeptides have been used as antibacterial agents against Gram-positive bacteria and anaerobes, including pathogens resistant to antibiotics currently in use [13, 14], and also have potential as growth inhibitors of the human malaria parasite [15]. They were discovered as antibiotics in diverse bacteria including Streptomyces, Bacillus,and Micrococcus [16, 17]. Thiopeptides have been proved later as effective growth promoters for domestic animals [17, 18]. Most of the thiopeptide antibiotics inhibit protein synthesis in bacteria and share a com- In our search for new antibiotics from terrestrial mon mode of action [19]. Thiostrepton, whose antibi- bacteria, the crude extracts of Streptomyces sp. iso- otic activity is best understood, acts by binding tightly late RSF18 showed a potent activity against the Gram- to the prokaryotic ribosome and thus inhibits trans- positive bacteria Bacillus subtilis, Staphylococcus au- lation [19, 20]. Geninthiocin (1) is known as an acti- reus and Streptomyces viridochromogenes (T¨u 57), be- vating agent for transcription of the tip A promoter in side a weak antifungal activity against Mucor miehei streptomycetes [21]. (T¨u 284) and Candida albicans. Extraction of the

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Fig. 1. CID-MS/MS spectrum of Val-geninthiocin (2); fragmentation of [M+H]+ measured on a quadrupole ion trap instrument. mycelia cake followed by a series of chromatographic extracts from water and mycelia, respectively, showed steps afforded two thiopeptides, geninthiocin (1)and a completely different metabolic pattern. Work-up of the new closely related Val-geninthiocin (2)astwocol- the mycelial extract using a silica gel column and size orless solids. Val-Geninthiocin (2), a desoxy-derivative exclusion chromatography resulted in geninthiocin (1; of geninthiocin (1), is an UV absorbing middle-polar 78.6 mg) and a second closely related peptide (2; substance, which turned yellow by spraying with 27.5 mg), as the NMR data indicated. Separation of anisaldehyde/sulfuric acid reagent. Extraction of the the filtrate extract delivered chalcomycin (19.8 mg) as filtrate afforded chalcomycin, a well-known macrolide a white solid. antibiotic [22]. HRMS of the main peptide isolated from Strepto- myces sp. RSF18 delivered the formula C50H49N15- Results and Discussion O15S, while the minor component had the composition C50H49N15O14S. A data base search pointed to Well-grown agar plates of strain RSF18 were used geninthiocin (1) for the former one, while the sec- to inoculate 60 of 1 L Erlenmeyer flasks, each con- ond product was new. As part of our ongoing struc- taining 250 mL of M2 medium. The fermentation was ture elucidation of cyclopeptides by MS methods, it carried out at 95 rpm on a linear shaker for 7 d was of interest to investigate the fragmentation be- at 28 ◦C, forming a yellowish brown culture broth. Af- havior of both metabolites in parallel. For this pur- ter harvesting, the broth was filtered over Celite, and pose, MS2 and MS3 experiments using a quadrupole the filtrate was adsorbed on Amberlite XAD-16 resin, ion trap were performed (Table 1), and additionally, followed by elution with methanol. After concentra- high-resolution CID-MS/MS measurements were car- tion, the aqueous residue was further extracted with ried out on a Fourier transform ion cyclotron resonance ethyl acetate followed by evaporation in vacuo, yield- (FT-ICR) mass spectrometer to determine the elemen- ing 1.30 g of crude extract. The mycelial cake was ex- tal composition of key fragments (Table 2). tracted with ethyl acetate followed by acetone; evap- Tandem mass spectrometry is a very powerful oration and re-extraction with ethyl acetate delivered method for sequence analysis of peptides and proteins. in a similar way 6.85 g crude extract. TLC of both The fragmentation of linear peptides was described I. Sajid et al. · Val-Geninthiocin 1225

Table 1. MS2 and MS3 product ions of the [M+H]+ precursor ions of geninthiocin (1) and Val-geninthiocin (2) obtained on a quadrupole ion trap mass spectrometer. Geninthiocin (1) Val-Geninthiocin (2) MS2 fragment ion MS3 fragment ions MS2 fragment ion MS3 fragment ions Proposed MS2 fragmention m/zm/zm/zm/z 1115 1099 –NH3 1114 1098 –H2O 1104 1088 –CO 1074 1056; 1046; 988; 936; –C3H6O from Hyval 907; 855; 812; 798 1046 1028; 1018; 985; 949; 1030 1012; 1002 –(H-Deala-NH2) 879; 851 1017 1000; 999; 982; 931; 1017 1000; 999; 931; 879; 798; –Hyval/Val 879; 862; 798; 579 579; 493 994 966; 936; 974; 370 978 960; 950; 370 –C6H6N2O2 from side chain 965 948; 947; 921; 907; 949 931; 921; 905; 903; 863; –(H-Oxa-NH2) 903; 879; 850; 827; 850; 811; 631 798; 631 947 931 –(H-Oxa-NH2)–H2O 913 896; 895; 877; 855; 897 879; 851; 833; 811; 798; –Oxa–Deala 852; 837; 798 793; 579 907 889; 879; 863; 821; –C10H15N3O3–(H-Oxa-NH2)–C3H6O 769; 752; 631 896 880 –H–Oxa–Deala–NH2 895 878; 877; 851 879 861; 851 –Oxa–Deala–H2 O 889 –C10H17N3O4–(H-Oxa-NH2)– C3H6O–H2O 852 850 850 –H–Hyval/Val–Oxa–NH2 798 781; 780; 770; 754; 798 781; 780; 770; 754; 712; –Hyval/Val–Oxa–Deala 712; 660; 631; 579 660; 631; 579 780 780 –Hyval/Val–Oxa–Deala–H2 O 746 729; 728; 660; 608; 579 746 729; 728; 660; (608; 579) –Deala–Oxa–Hyval/Val–COCCH2 +2H 631 603; 545; 493 631 603; 545; 493 –H–Oxa–Deala–Hyval/Val–Oxa–NH2 579 551; 535; 493; 441 579 561; 551; 535; 493; 441 –Oxa–Deala–Hyval/Val–Oxa–Deala 508 370 508 370

Table 2. Conﬁrmation of key fragments by exact mass deter- However, the structure analysis of cyclic peptides, mination using a FT-ICR mass spectrometer. which represent an important class of bioactive natu- Mass Composition Mass ∆m Fragmentation ral products, by mass spectrometry remains a challeng- (measd.) (calcd.) (ppm) ing task. Complex fragmentation patterns by two-bond Geninthiocin (1) cleavage at different ring positions, ring-opening reac- + 965.2619 C43H41N12O13S1 965.2631 1.3 [M+H–C7H9N3O2] + tions and uncommon rearrangement reactions compli- 947.2523 C43H39N12O12S1 947.2526 0.2 [M+H–C7H11N3O3] + cate the interpretation of CID-MS/MS spectra [25, 26]. 907.2203 C40H35N12O12S1 907.2213 1.1 [M+H–C10H15N3O3] + n 889.2102 C40H33N12O11S1 889.2107 0.6 [M+H–C10H17N3O4] Frequently, higher-order MS investigations are re- + 798.2040 C35H32N11O10S1 798.2049 1.1 [M+H–C15H18N4O5] quired. Furthermore, cyclic peptides produced as sec- + 746.2095 C32H32N11O9S1 746.2100 0.7 [M+H–C18H18N4O6] + ondary metabolites by bacteria often contain uncom- 631.1348 C28H23N8O8S1 631.1354 0.9 [M+H–C22H27N7O7] mon amino acid residues leading to different fragmen- Val-Geninthiocin (2) tation reactions. + 949.2678 C43H41N12O12S1 949.2682 0.4 [M+H–C7H9N3O2] + In general, for sequencing linear peptides, fragmen- 931.2577 C43H39N12O11S1 931.2576 0.1 [M+H–C7H11N3O3] 2+ + tation of the [M+2H] ions is used successfully, but 897.2723 C40H41N12O11S1 897.2733 1.1 [M+H–C10H9N3O3] + 798.2036 C35H32N11O10S1 798.2049 1.6 [M+H–C15H18N4O5] in the case of geninthiocin (1) and its minor con- + 631.1359 C28H23N8O8S1 631.1354 0.8 [M+H–C22H27N7O6] gener, fragmentation spectra of those ions were dominated by doubly charged fragment ions without signif- comprehensively, and detailed knowledge of the frag- icant sequence information. Furthermore, [M+2H]2+ mentation mechanism has been obtained [23, 24]. ions were observed with very low intensity under 1226 I. Sajid et al. · Val-Geninthiocin

III. Cleavage of Oxa-Deala dipeptide units with high preference resulted in the prominent fragment ion peaks at m/z = 913 (1) and 897 (2), respectively. Fur- thermore, fragmentation of Deala-Hyval/Val occurred with lower intensity. IV. Cleavage of the tripeptide unit Hyval/Val-Oxa- Deala gave the key fragment at m/z = 798. After cleavage of the tripeptide unit Hyval/Val-Oxa-Deala, a further dipeptide Oxa-Deala loss resulted in the fragment ion m/z = 579 (IV+III). V. Fragmentation of the linear peptide unit predom- inantly occurred by cleavage of the NH–CO bond between Deala(4) and Deala(3) forming a b-fragment ion (Vb10). Furthermore, formation of a c-type fragment (Vc9)bycleavageofaCH2=C–NH bond between Deala(3) and Pyr induced by the dehydroalanine structure was observed. The fragmentation pathways derived from collision activated dissociation using a quadrupole ion trap (Ta- Fig. 2. Selected CID-MS/MS key fragments of the [M+H]+ ble 1) were confirmed by high-resolution MS/MS in precursor ions of geninthiocin (1, R = OH) and Val- an FT-ICR mass spectrometer providing the elemental geninthiocin (2, R = H) obtained on a quadrupole ion trap composition of key fragments (Table 2). By compar- mass spectrometer; for detailed information, see Table 1. ison of MS2 spectra, the position of valine in 2 was determined unambiguously. The information obtained the conditions described. Also the MS2 spectra of + − on fragmentation pathways can be applied in further [M+Na] and [M–H] provided little sequence in- investigations on this class of substances to identify formation. Therefore, CID-MS/MS studies were con- + small amounts of derivatives. centrated on the more abundant [M+H] species The 1Hand13C NMR spectroscopic data of (Fig. 1). geninthiocin (1) and Val-geninthiocin (2) are summa- Beside an unspecific loss of H2O, CO, and NH3,the rized in Table 3. The 13C NMR spectrum of Val- following main fragmentation pathways were observed geninthiocin (2) revealed the presence of 50 carbon (Latin numbers correspond to the fragments indicated atoms as for geninthiocin (1), in accordance with the in Fig. 2): empirical formula. While the 1H NMR spectrum of 2 I. Cleavage of a single amino acid residue unit from showed 49 protons, of which 12 were D2O exchange- the peptide ring system was found basically for Hy- able, geninthiocin (1) exhibited 13 exchangeable pro- val (−115 Dalton) in 1 and Val (−99 Dalton), respec- tons. This pointed to one hydroxy group less in 2,in tively, for 2 (cleavage of two CO–NH peptide bonds). accordance with the empirical formula. The respective Therefore, the structural difference between both com- hydroxy signal (δ = 5.17) in geninthiocin (1)wasre- pounds could be allocated at this amino acid at a very placed in 2 by the methine hydrogen signal of an iso- early stage. propyl unit. Accordingly, the singlets of the geminal II. Cleavage of a single amino acid residue unit dimethyl group of hydroxyvaline in geninthiocin (1) from the peptide ring system by fragmentation of at δ = 1.22 and 1.20 were substituted in 2 by a 6H one CO–NH peptide bond and one CH2=C–NH bond doublet (δ = 0.97, J ∼ 6.6 Hz), whose coupling part- (H-Oxa-NH2, H-Hyval/Val-NH2, H-Deala-NH2)was ner gave a multiplet at δ = 2.20. An isobutyl fragment 13 detected. Obviously, the -NH–C=CH2- bond between was further confirmed by the C NMR spectra of 2, Oxa(1)-Deala(1) and Oxa(3)-Deala(2) as well as be- which exhibited the replacement of the oxygenated β- tween Deala(2)-Oxa(2) and Hyval/Val-Oxa(3) can be carbon signal (δ = 71.0) of hydroxyvaline in 1 by a broken favorably leading to a second preferred frag- methine signal (δ = 29.5) in compound 2. Besides the mentation pathway beside peptide bond (-CO–NH-) values of hydroxyvaline, all other shifts were nearly cleavage. identical with those of 1. The NMR data confirmed I. Sajid et al. · Val-Geninthiocin 1227

13 1 Table 3. Cand H NMR data of geninthiocin (1) and Val-geninthiocin (2)in[D6]DMSO in comparison with literature data. Unit Position Geninthiocin (1) (lit. data) [21] Geninthiocin (1) (exp. data) Val-Geninthiocin (2) a b a b δC δH (J in Hz) δC δH (J in Hz) δC δH (J in Hz) Thiazole C-2 163.2 – 163.1 – 163.0 – C-4 149.4 – 149.4 – 149.4 – CH-5 126.9 8.49 126.7 8.49 (s) 126.6 8.49 CO 159.9 – 159.9 – 159.8 – Threonine NH – 8.00 (d, 9.0) – 8.00 (d, 9.0) 8.00 (d, 9.2) αCH 57.8 4.60 (dd, 9.0, 3.0) 57.7 4.60 (dd, 9.0, 3.0) 57.6 4.60 (dd, 9.0, 3.0) βCH 67.3 4.29 (m) 67.2 4.29 (m) 67.3 4.29 (m) γCH3 20.5 1.14 (d, 6.2) 20.4 1.14 (d, 6.2) 20.3 1.14 (d, 6.1) OH – 4.98 (d, 5.5) – 4.98 (d, 5.5) – 4.95 (d, 5.4) CO 168.8 – 168.7 – 168.6 Oxazole (1) NH – 9.60 – 9.55 (s) – 9.60 (s br) αC 123.1 – 123.1 – 123.0 – βCH 129.5 6.55 (q, 7.3) 129.5 6.55 (q, 7.3) 129.2 6.53 (q, 7.0) γCH3 13.8 1.74 (d, 7.3) 13.7 1.74 (d, 7.3) 13.6 1.75 (d, 7.0) C-2 159.4 – 159.4 – 159.4 – C-4 136.1 – 136.0 – 136.1 – CH-5 142.7 8.70 142.6 8.70 (s) 142.5 8.70 (s) CO 158.4 – 158.3 – 158.3 – Dehydroalanine (1) NH – 9.39 – 9.39 (s) – 9.40 (s br) αC 133.4 – 133.4 – 133.3 – βCH2 103.7 6.46, 5.88 103.7 6.46, 5.88 105.8 6.46 (s), 5.88 (s) CO 163.7 – 163.7 – 163.7 – Hydrovaline and Valine NH – 8.23 (d, 8.5) 8.23 (d, 8.5) 8.25 (d, 8.1) αCH 61.8 4.64 (d, 8.5) 61.7 4.64 (d, 8.5) 60.0 4.61 (dd, 8.8, 2.8) βC 71.0 – 71.0 – 29.5 2.20 (m) γCH3 27.3 1.22 27.3 1.22 (s) 18.9 0.97 (d, 6.6) γCH3 26.1 1.20 26.0 1.20 (s) 18.8 0.97 (d, 6.6) OH – 5.17 – 5.17 (s) – – CO 169.4 – 169.3 – 171.0 – Oxazole (3) NH – 9.61 – 9.60 – 9.60 αC 128.5 – 128.5 – 128.6 – βCH2 105.6 6.13, 5.65 105.7 6.13, 5.65 106.1 6.09, 5.70 C-2 155.2 – 155.1 – 155.1 – C-4 129.2 – 129.2 – 129.2 – C-5 154.6 – 154.5 – 154.2 – CH3-5 11.5 2.62 11.5 2.62 11.4 2.63 (s) CO 159.5 – 159.5 – 159.4 – Dehydroalanine (2) NH – 9.36 – 9.36 – 9.37 (s br) αC 133.9 133.9 133.9 βCH2 105.9 6.36, 5.76 105.9 6.36, 5.76 105.8 6.36, 5.76 CO 162.7 162.7 162.6 Oxazole (2) NH – 9.77 – 9.77 – 9.69 αC 129.3 129.1 130.0 ∗ βCH2 111.2 5.70, 5.71 111.0 5.70, 5.71 111.2 5.70, 5.73 C-2 158.1 – 158.3 – 158.0 – C-4 139.2 – 139.2 – 139.1 – CH-5 140.5 8.68 140.5 8.68 140.4 8.68 (s) Pyridine C-2 149.2 – 149.2 – 149.2 – C-3 130.2 – 130.1 – 130.1 – CH-4 141.0 8.50 (d, 8.0) 141.0 8.50 (d, 8.0) 140.8 8.52 (d, 8.2) CH-5 121.5 8.23 (d, 8.0) 121.3 8.23 (d, 8.0) 121.4 8.25 (d, 8.1) C-6 146.8 – 146.8 – 146.8 – CO 161.6 – 161.6 – 161.5 – Dehydroalanine (3) NH – 10.53 – 10.53 – 10.51 (s br) αC 134.7 – 134.7 – 134.7 – ∗ βCH2 106.2 6.42, 5.83 106.1 6.42, 5.83 106.0 6.43, 5.84 CO 162.0 – 162.0 162.0 Dehydroalanine (4) NH – 9.44 – 9.44 – 9.42 (s br) αC 135.1 – 135.0 – 135.0 – βCH2 106.0 6.03, 5.69 106.0 6.03, 5.69 106.0 6.05, 5.70 CO 165.1 – 165.1 – 165.0 – NH2 – 7.93, 7.50 – 7.93, 7.50 – 7.92, 7.48 a (75 MHz); b (300 MHz); ∗ expected value. 1228 I. Sajid et al. · Val-Geninthiocin the MS-derived structure 2, for which we suggest the Table 4. Antibacterial and antifungal activities of name Val-geninthiocin. According to the NMR and OR geninthiocin (1) in comparison with Val-geninthiocin (2) and chalcomycin (conc. 40 µgperdisk). data, our 1-sample had the same absolute configuration as reported for geninthiocin in the literature [21]; the Tested microorganisms Inhibition zone ∅ (mm) same configuration is plausible also for 2. Genin- Val-Genin- Chalco- thiocin (1) thiocin (2)mycin Bacillus subtilis 15 13 29 Biological activities Staphylococcus aureus 15 14 36 Streptomyces virido- 14 11 37 Biological activities of geninthiocin (1), Val- chromogenes (Tü 57) geninthiocin (2) and chalcomycin were measured us- Escherichia coli ––– Candida albicans 11 11 11 ing the agar diffusion method. In comparison with 1, Mucor miehei (Tü 284) 11 11 11 compound 2 showed slightly lower antibacterial ac- Chlorella vulgaris ––– tivities against Gram-positive bacteria, viz. Bacillus Chlorella sorokiniana ––– subtilis, Staphylococcus aureus and Streptomyces viri- Scenedesmus subspicatus ––– dochromogenes (Tü 57), and minor antifungal activity against Mucor miehei (Tü 284) and Candida albi- Accompanying high-resolution MS/MS investigations cans. Both compounds exhibited, however, no activi- were performed on an Apex IV 7 Tesla Fourier-Transform ties against the Gram-negative Escherichia coli and the Ion Cyclotron Resonance Mass Spectrometer. Samples were infused with a flow rate of 2 µL/min. CID-MS/MS studies microalgae Chlorella vulgaris, Chlorella sorokiniana + were carried out on the [M+H] species inside the ICR cell and Scenedesmus subspicatus (Table 4). with argon as collision gas. Conditions: electrospray voltage 4.2 kV, capillary exit voltage 100 V, nebulizing gas ni- Experimental Section trogen (30 psi), drying gas nitrogen (250 ◦C), hexapole accumulation 0.1 s, mass range m/z = 100 – 1400. Optical rotations: Polarimeter (Perkin-Elmer, model 243). UV/Vis spectra were recorded on a Perkin-Elmer Lambda 15 Taxonomy of the producing strain UV/Vis spectrometer. NMR spectra were measured on Var- ian Unity 300 and Varian Inova 600 spectrometers. Elec- The Streptomyces sp. RSF18 was isolated from the soil trospray ionization mass spectrometry (ESI-MS): Finnigan of a rose field at the province Punjab, Pakistan. The tax- LCQ ion trap mass spectrometer. High-resolution mass spec- onomic status of the strain RSF18 was determined by pre- tra (HRMS) were recorded by ESI MS on an Apex IV 7 liminary physiological testing and 16S rRNA gene sequenc- Tesla Fourier-Transform Ion Cyclotron Resonance Mass ing. Cultural characteristics were observed during the incu- Spectrometer (Bruker Daltonics, Billerica, MA, USA). Size bation at 27 ◦C for 21 d on GYM medium [27]. It formed a exclusion chromatography was done on Sephadex LH-20 pale yellowish substrate mycelium, which changed to dark (Pharmacia). Rf values were measured on a Polygram SIL brown after 14 d of incubation. The aerial mycelium was F/UV254 system (Merck, pre-coated sheets). powdery and characteristically whitish to grey in color on prolonged incubation. Melanoid pigment was produced, and MS/MS studies a soluble, slightly orange pigment was formed. For the cultural characteristics, physiological properties, and utilization Samples of geninthiocin (1) and Val-geninthiocin (2)were of carbon sources of strain RSF18 see Table 5. Permissive dissolved in methanol/water (75/25) containing 0.1 % formic temperature ranges for growth of the strain RSF18 were 20 acid. Low resolution MS2 and MS3 measurements were per- to 37 ◦C with an optimum at 28 ◦C. Comparison of these formed on an LCQ quadrupole ion trap instrument (Finni- characteristics with those of actinomycete species described gan, San Jose, USA) using electrospray ionization in the pos- in Bergey’s Manual of Systematic Bacteriology [28] strongly itive and negative ionization mode with an electrospray volt- suggested that strain RSF18 belongs to the genus Strep- age of +/− 4.5 kV. Samples were introduced by means of tomyces. Taxonomic determination, PCR amplification and a syringe pump with a flow rate of 3 µL/min. The capil- 16S rRNA gene sequencing were performed as described lary was heated to 200 ◦C. [M+H]+,[M+2H]2+,[M+Na]+, previously [29]. The nucleotide sequence of 1418 bp (ac- and [M–H]− ions were isolated (isolation width 3.0 Dalton) cession number EU294139) of the 16S rRNA gene of the and submitted to fragmentation by collision-induced disso- Streptomyces sp. RSF18 was determined in both strands. The ciation with helium as collision gas. For MS3 investigations alignment of this sequence through matching with reported fragment ions were isolated (isolation width 3.0 Dalton peak 16S rRNA gene sequences in the gene bank showed high width) and fragmented under the same conditions. similarity (98 – 99 %) to Streptomyces 16S rRNA genes. I. Sajid et al. · Val-Geninthiocin 1229

RSF18 (15 L Shaker culture)

7 days

mixed with Celite and filtered by filterpress Biomass Filtrate 4 x EtOAC & XAD-16 1x Acetone (MeOH/H2O), EtOAC Crude extract Crude extract (6.85 g) (1.30 g)

SCC (CH Cl -MeOH) 2 2 SCC (CH2Cl2-MeOH)

B2, A1

Fat A2, B1, A1 Fat Chalcomycin

A1 = Sephadex LH-20 (CH2Cl2/40% MeOH)

A2 = Sephadex LH-20 (CH2Cl2/50%MeOH) Fig. 3. Work-up scheme of B1 = PTLC (CH Cl /6 % MeOH) Geninthiocin (1) 2 2 the terrestrial Streptomyces sp. Val-Geninthiocin (2) B2 = PTLC (CH Cl /5 % MeOH) 2 2 RSF18.

Table 5. Cultural and physiological characteristics of strain used to inoculate 60 of 1 L Erlenmeyer flasks each contain- a RSF18 . ing 250 mL of M2 medium, and further incubated for 7 d ◦ Growth pattern well grown ∼ partitioned as shaker culture (95 rpm) at 28 C. The resulting yellow- Substrate mycelium yellowish ∼ dark brown ish brown culture broth was mixed with ca. 1 kg diatoma- Ariel mycelium whitish ∼ grey ceous earth, pressed through a pressure filter giving filtrate ∼ ◦ Growth temperature range 20 37 C and biomass, which were extracted separately. The filtrate Production of melanin + Utilization of organic acids + was subjected to adsorption on Amberlite XAD-16, and the Utilization of oxalates + resin subsequently extracted with methanol. The methanol Hydrolysis of urea + extract was concentrated, and the resulting aqueous residue Hemolysis − was re-extracted with ethyl acetate followed by evaporation Carbon source utilization in vacuo to dryness, affording 1.30 g of brown extract. The D-Glucose + mycelia phase was extracted 3 times with ethyl acetate fol- L-Arabinose + lowed by acetone (1 time). The acetone was evaporated from Sucrose − the aqueous residue, which was finally extracted with ethyl D-Fructose + acetate. The combined ethyl acetate extracts were concen- + D-Mannitol trated in vacuo to dryness giving 6.85 g of brown extract. Raffinose + − D-Galactose Isolation Soluble starch + Glycerol + Fractionation and purification of the mycelia extract a +: Positive; −: negative. (6.85 g) using silica gel column chromatography and elution with a CH2Cl2/MeOH gradient gave two crude fractions of 1 M2 medium and 2. Further purification by PTLC and Sephadex LH-20 resulted in two colorless solids of geninthiocin (1; 78.6 mg) A solution of 10 g malt extract, 5 g yeast extract and and Val-geninthiocin (2; 27.5 mg). Chromatography of the 5 g glucose in 1 L of tap water was set to pH = 7.8 with ◦ filtrate extract (1.3 g) using silica gel with a CH Cl -MeOH 2 N NaOH and sterilized for 30 min at 121 C. 2 2 gradient followed by PTLC and Sephadex LH-20 (see Fig. 3) delivered chalcomycin as a white solid (19.8 mg). Fermentation of isolate RSF18 Geninthiocin (1) The terrestrial isolate Streptomyces sp. RSF18 was inoculated from its storage culture on three M2 agar plates and in- White amorphous solid. Yellow with anisaldehyde/sul- cubated for 96 h at 28 ◦C. The well-developed colonies were furic acid spraying reagent. Soluble in DMSO, MeOH, 1230 I. Sajid et al. · Val-Geninthiocin

EtOH and EtOAc. Insoluble in hexane and CH2Cl2.– MeOH, EtOH and EtOAc. Insoluble in hexane and 1 1 Rf =0.55(CH2Cl2/10 % MeOH). – H NMR ([D6]DMSO, CH2Cl2.–Rf =0.61(CH2Cl2/10 % MeOH). – HNMR 13 13 300 MHz) and C NMR ([D6]DMSO, 75 MHz): see Ta- ([D6]DMSO, 300 MHz) and C NMR ([D6]DMSO, [α]25 + [α]25 + ble 3. – D = 155 (c = 0.1, MeOH). – UV/Vis (MeOH): 75 MHz): see Table 3. – D = 131 (c =0.1, λmax(lgεmax) = 237 nm (5.09); (MeOH/HCl): 238 nm MeOH); UV (MeOH): λmax(lgεmax) = 237 nm (4.93); (5.04); (MeOH/NaOH): 238 nm (5.04). – (+)-ESIMS: m/z = (MeOH/HCl): 239 (4.92), 205 nm (4.94); (MeOH/NaOH): 1154.3 [M+Na]+.–(−)-ESIMS: m/z = 1130.2 [M–H]−.– 239 nm (4.92). – (+)-ESIMS: m/z = 1138.4 [M+Na]+.– (+)-HRESIMS: m/z = 1132.33319 (calcd. 1132.33260 for (+)-HRESIMS: m/z = 1116.33707 (calcd. 1116.33769 for + + C50H50N15O15S, [M+H] ), 1154.31444 (calcd. 1154.31455 C50H50N15O14S, [M+H] ), 1138.31812 (calcd. 1138.31963 + + for C50H49N15O15SNa, [M+Na] ). for C50H49N15O14SNa, [M+Na] ). Acknowledgements Val-Geninthiocin (2) We thank Mr. R. Machinek for NMR measurements, White amorphous solid. Yellow with anisalde- F. Lissy for biological activity tests and A. Kohl for technical hyde/sulfuric acid spraying reagent. Soluble in DMSO, assistance.

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I3-O-008 I3-O-012

Val-Geninthiocin: A thiopeptide antibiotic produced by Strepto- Investigating the action mechanism for a natural active com- myces sp. RSF18 pound honokiol by quantitative proteomics

Imran Sajid 1,2,∗, Khaled A. Shaaban 1, Holm Frauendorf 1, Shahida Shufang Liang 1,∗, Yuhuan Xu 1, Bo Ling 2, Xia Zhao 2, Jin Zhou 1, Hasnain 2, Hartmut Laatsch 1 Lijuan Chen 1, Yuquan Wei 1

1 Institute of Organic and Biomolecular Chemistry, Tammanstrasse 2, 1 State Key Laboratory of Biotherapy, West China Hospital, Sichuan Georg-August University of Gottingen 37077, Germany University, Chengdu 610041, China 2 Department of Microbiology and Molecular Genetics, University of 2 West China Second Hospital, Sichuan University, Chengdu 610041, the Punjab, Quaid-e-Azam Campus, Lahore 54590, Pakistan China

Thiopeptide antibiotics are forming a group of cyclic peptides char- E-mail address: [email protected] (S. Liang). acterized by several common structural features, such as oxazole The quantitative proteomics brings about a new method to inves- and thiazole units and unusual amino acids; especially dehy- tigate the action mechanism of natural active compounds from droamino acids are typical (Chiu et al., 1999). Thiopeptides have organisms. The SILAC (stable-isotope labelling by amino acids in cell been used as antibacterial agents against Gram-positive bacteria culture) combined with mass spectrometry (MS) has emerged as a and anaerobes, including pathogens resistant to antibiotics cur- simple and powerful quantitative proteomic technique (Ong et al., rently in use (Nagai et al., 2003), and also have potential as growth 2002). Honokiol (HNK), an active component purified from Magno- inhibitors of the human malaria parasite (Rogers et al., 1998). Val- lia officinalis, a plant used in traditional Chinese, exhibits antitumor Geninthiocin (1), a new member of thiopeptide antibiotics, was effects by inhibiting tumor growth (Bai et al., 2003), while proteins isolated from the mycelium of Streptomyces sp. RSF18, along with involved in antitumor activity in proteomic level are still unclear. In the closely related geninthiocin (2) and the macrolide, chalcomycin. our study, HNK could inhibit cell proliferation and induce apoptosis Compound 1 showed potent activity against Gram-positive bacte- of HeLa and HepG2 cells in a concentration- and time-dependent ria and minor antifungal activity, while it was not effective against manner. We applied the SILAC–MS technique to analyze the dif- Gram-negative bacteria or microoalgae. By intensive NMR and MS ferential proteome profiling of cells treated by HNK to investigate studies, structure of 1 was established as a thiopeptide contain- key proteins responsible for HNK activities. The changed proteins ing several oxazole and thiazole units and a number of unusual covered a broad variety of cellular functions including metabolism, amino acids. We are describing here the fermentation, isolation signal transduction etc., which indicated HNK performs cytotoxicity and structure elucidation as well as the biological activity of 1. to tumor cells through co-operating of many proteins and different pathways. Among these changed proteins, IQGAP1, ␤-tubin, Keywords: Val-Geninthiocin; Thiopeptide antibiotic; Streptomyces peroxiredoxin-6 and HSP70 etc. proteins were down-regulated significantly, while proteins including annexin A2 etc. were up- References regulated after HNK treatment. Since IQGAP1 plays important roles in cell adhesion and migration (Noritake et al., 2005), we supposed Chiu, M.L., Folche, M., Katoh, T., Puglia, A.M., Vohradsky, J., Yun, B.S., Seto, H., Thompson, C.J., 1999. Broad spectrum thiopeptide recognition specificity of the that HNK may have effects on cell migration through IQGAP1 based Streptomyces lividans TipAL protein and its role in regulating gene expression. J. on our MS datasheet. Our further scratch migration assay showed Biol. Chem. 274, 20578–20586. that the migration inhibition of HepG2 cells can be induced by HNK, Nagai, K., Kamigiri, K., Arao, N., Suzummura, K., Kawano, Y., Yamaoka, M., Zhang, H., Watanabe, M., Zuzki, K., 2003. YM-266183 and YM-266184, novel thiopep- the RNA and protein expression level of IQGAP1 were respectively tide antibiotics produced by Bacillus cereus isolated from a Marine Sponge. I. decreased obviously in HepG2 cells exposed to 10 ␮g/ml HNK for Taxonomy, fermentation, isolation, physico-chemical properties and biological 24 h. Therefore, the down-regulated expression of IQGAP1 by HNK properties. J. Antibiot. 56, 123–128. Rogers, M.J., Cundliffe, E.T., McCutchan, F., 1998. The antibiotic micrococcin is a treatment was correlated with cell migration, and HNK probably potent inhibitor of growth and protein synthesis in the malaria parasite. Antimi- inhibits cell proliferation and migration through IQGAP1 expression crob. Agents Chemother. 42, 715–716. changes and its interactions with other proteins. doi:10.1016/j.jbiotec.2008.07.169 References

Bai, X., Cerimele, F., Ushio-Fukai, M., Waqas, M., Campbell, P.M., Govindarajan, B., Der, C.J., Battle, T., Frank, D.A., Ye, K., Murad, E., Dubiel, W., Soff, G., Arbieser, J.L., 2003. Honokiol, a small molecular weight natural product, inhibits angiogenesis in vitro and tumor growth in vivo. J. Biol. Chem. 278, 35501–35507. Noritake, J., Watanabe, T., Sato, K., Wang, S., Kaibuchi, K., 2005. IQGAP1: a key regu- lator of adhesion and migration. J. Cell Sci. 118, 2085–2092. Ong, S.E., Blagoev, B., Kratchmarova, I., Kristensen, D.B., Steen, H., Pandey, A., Mann, M., 2002. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell. Proteomics 1, 376–386.

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∗ Corresponding author at: Institute of Organic and Biomolecular Chemistry, Tam- manstrasse 2, Georg-August University of Gottingen 37077, Germany. Abstracts / Journal of Biotechnology 136S (2008) S75–S98 S79 dants. There are a number of synthetic chemical routes to making sition analysis of the volatile oil from endophytic P. guilliermondii a range of variously substituted alkyltriphenylphosphonium com- isolated from P. polyphylla var. yunnanensis and its antibacterial pounds. We used the TPP cation as the primary carrier for the initial activity which indicated that endophytic P. guilliermondii volatile work in examining the possibility of targeting antioxidants to mito- oil may have a potential as an antimicrobial agent in agricultural chondria. Curcumin, a natural product of turmeric has been claimed and pharmaceutical applications. to work as an excellent antioxidant (Balasubramanyam et al., 2003). Such mitochondria-targeted antioxidants have been developed by conjugating the lipophilic triphenylphosphonium cation to an Acknowledgements antioxidant moiety, such as ubiquinol or ␣-tocopherol. These compounds pass easily through all biological membranes, including the This work was co-financed by the National Key Technology blood–brain barrier, and into muscle cells and thus reach those R&D Program of China (2007BAD57B02 and 2006BAD08A03) and tissues most affected by mitochondrial oxidative damage. It was National Natural Science Foundation of China (30470038). then validated for its beneficial actions in tissue culture and animal models like rat with special reference to counteracting oxidative References stress under hyperglycemia conditions. Thus ‘MITO-CURCUMIN’ (our brain child) is developed and it has a number of ‘translational Adams, R.P., 2001. Identification of Essential Oils Components by Gas Chromatogra- applications’. It will scientifically position our natural product in the phy/Quadrupole Mass Spectroscopy. Allured Publishing Corporation, USA. international bioprospecting market and increase our economy. Langfied, R.D., Scarano, F.J., Heitzman, M.E., Kondo, M., Hammond, G.B., Neto, C.C., 2004. Use of a modified microplate bioassay method to investigate antibacterial activity in the Peruvian medicinal plant Peperomia galioides. J. Ethnopharmacol. 94, 279–281. References

Balasubramanyam, M., Adaikalakoteswari, A., Sampathkumar, R., Mohan, V., 2003. doi:10.1016/j.jbiotec.2008.07.177 Curcumin-induced inhibition of cellular reactive oxygen species (ROS) generation: novel therapeutic implications. J. Biosci. 28, 715–721. Ceriello, A., Motz, E., 2004. Is oxidative stress the pathogenic mechanism underly- I3-P-009 ing insulin resistance, diabetes, and cardiovascular disease? The common soil hypothesis revisited. Arterioscler. Thromb. Vasc. Biol. 24 (5), 816–823. Antifungal and antibacterial activities of indigenous Strepto- myces isolates from saline farmlands, prescreening, ribotyping doi:10.1016/j.jbiotec.2008.07.176 and metabolic diversity

Imran Sajid 1,2, Clarisse Blandine Fotso Fondja Yao 2, Khaled Attia I3-P-004 Shaban 2, Shahida Hasnain 1,∗, Hartmut Laatsch 2

Composition analysis of the volatile oil from endophytic fungus 1 Department of Microbiology and Molecular Genetics, University of Pichia guilliermondii from Paris polyphylla var. Yunnanensis and the Punjab, Quaid-e-Azam Campus, Lahore 54590, Pakistan its antibacterial activity 2 Institute of Organic and Biomolecular Chemistry, Tammanstrasse 2, Georg-August University of Gottingen 37077, Germany Jianglin Zhao, Yongfu Huang, Lijian Xu ∗, Xiaoyue Cai, Zhanhong Ma, Ligang Zhou The antibiotic resistance of a large number of pathogenic bacteria and fungi is presently an urgent focus of research, and new anti- Department of Plant Pathology, College of Agronomy and Biotechnol- fungal and antibacterial molecules are necessary to combat these ogy, China Agricultural University, Beijing 100193, China pathogens (Fguira et al., 2005). Soil, in particular, is an intensively E-mail address: [email protected] (L. Xu). exploited ecological habitat, the microorganisms of which produce A volatile oil from aroma producing fungal endophyte Pichia guil- many useful natural products, including clinically important antibi- liermondii Ppf9 (accession number EF495244 in GenBank) isolated otics (Waksman, 1967). In the screening for new bioactive microbial from rhizomes of Paris polyphylla var. yunnanensis (Franch) Hand compounds, three major factors must be considered, i.e., selection (Trilliaceae) was analyzed by gas chromatography–mass spec- of producing organisms, culture methods and detection of metabo- trometry (GC–MS). Twenty-seven components of the volatile oil lites. The screening process from isolation of the producing microbe were characterized by comparison of their mass spectra with those to recognition of a new active metabolite, earlier took up to one of NIST 2002 library data of the GC–MS system as well as their year (Berdy, 1985), but has shrunken to a few days, using elevated retention indices (RI; Adams, 2001). Eight main components were methods (Laatsch, 2000). A culture collection of about 110 indige- identified as 1,1,3a,7-tetramethyl-1a,2,3,3a,4,5,6,7b-octahydro-1H- nous Streptomyces strains originally isolated from saline farmlands cyclopropa[a] naphthalene (25.90%); palmitic acid (15.51%); using stringent methods was screened biologically and chemically (9E,12E)-ethyloctadeca-9,12-dienoate (9.20%); 1-methyl-2,4- to investigate their potential for the production of bioactive sec- di(prop-1-en-2-yl)-1-vinylcyclohexane (7.91%); (E)-octadec-9- ondary metabolites. In a biological screening the crude extracts enoic acid (7.28%); 2,3,4,5,6,6a,7,8-octahydro-3,6a,10-trimethyl- obtained from the culture broth of selected strains were analyzed 4,10a-methano-10aH-1-benzoxocin(4.81%);oct-1-en-3-ol (4.21%) for their activity against a set of test organisms including, Gram pos- and (4R, 4aS, 7aR, 7bR)-1,1,4,7-tetramethyl-decahydro-1H- itive bacteria, Gram negative bacteria, fungi and microalgae using cyclopropa[e]azulen-4-ol (3.44%). A modified broth dilution-MTT disc diffusion bioassay method, additionally a cytotoxicity test was assay (Langfied et al., 2004) was used to detect the minimal performed using Brine shrimp microwell cytotoxicity assay. In a inhibitory concentration (MIC) of the volatile oil against two gram- chemical screening each of the crude extract was analyzed by TLC positive (Bacillus subtilis and Staphylococcus haemolyticus) and four (thin layer chromatography) and HPLC–MS-MS measurements. A gram-negative bacteria (Escherichia coli, Xanthomonas vesicatoria, data base search for the measured masses provided the idea of the Agrobacterium tumefaciens and Pseudomonas lachrymans). MIC diversity of the chemical constituents of the crude extracts pro- values of P. guilliermondii oil ranged from 0.6 to 1.5 mg/ml. Maxi- duced by these strains. The taxonomical status of the strains was mum activity of the oil was obtained on X. vesicatoria that had the confirmed by preliminary biochemical analysis and 16s rRNA gene lowest MIC value (0.4 mg/ml). This is the first report on compo- sequencing. S80 Abstracts / Journal of Biotechnology 136S (2008) S75–S98

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