Thesis Sci 2007 Kirby B M.Pdf

The copyright of this thesis vests in the author. No quotation from it or information derived from it is to be published without full acknowledgementTown of the source. The thesis is to be used for private study or non- commercial research purposes only. Cape Published by the University ofof Cape Town (UCT) in terms of the non-exclusive license granted to UCT by the author. University The characterisation of actinomycetes isolated from diverse South African sources, with emphasis on the genus Kribbella by Town Cape Bronwyn Michelle Kirby of University Thesis Presented for the Degree of DOCTOR OF PHILOSOPHY in the Department of Molecular and Cell Biology F acuity of Science UNIVERSITY OF CAPE TOWN August 2007 Acknowledgements I wish to thank my supervisor Dr Paul Meyers for the guidance, insight and motivation he provided for the duration of this project. I am grateful for his endless patience while writing my thesis. I wish to thank Di James for teaching me so much about DNA sequencing, Miranda Waldron for assistance with SEM, and Associate Professors Shez Reid and Val Abratt for the use of the anaerobic chamber. I am grateful to the Medical Research Council, National Research Foundation, the Ernst & Ethel Eriksen Trust, the Postgraduate Studies Funding Committee and the Postgraduate Association, UCT, for personal financial assistance. I would like to thank my Dad and Mom for all their support and for allowing me to follow my heart. Thanks to my Gran for her wisdom. Special thanks to my sister, Kerry, for all her help and encouragement. I would like to thank Marilize for her knowledge, friendship and for sharing my amazement in these 'handsome' bugs. I would like to thank past and present lab members, especially Jeffrey,Town Gareth and Andrew, for all their help and for making the lab such a great place. Thanks to Rene, Amy and the rest of Lab 200 for their friendship, teatime jokes and Friday 'lab meetings'. Lastly, I would also like to thank Sergio for his support and friendship, even in his absence. Cape of University Table of Contents Abstract II Abbreviations IV Chapter 1 Introduction 1 Chapter 2 Characterisation of actinomycetes isolated from soil, 63 sediment and indigenous plant species Chapter 3 Screening of actinomycete isolates for antimicrobial 133 compounds with emphasis on antimycobacterial activities Chapter 4 The application of the gyrB gene to resolve species 173 relationships within the genus Kribbella Chapter 5 General Discussion Town 199 Cape of University II The characterisation of actinomycetes isolated from diverse South African sources, with emphasis on the genus Kribbella by Bronwyn Michelle Kirby August 2007 Abstract Actinomycetes were isolated from the leaves of indigenous plants, aquatic sediment and soil samples, using alternative isolation methods to select for actinomycetes belonging to the rarer genera. Thirty actinomycete strains belonging to the genera Gordonia, Kineococcus, Kribbella, Micromonospora,Town Nocardia and Streptomyces were selected for full characterisation. A polyphasic approach combining physiology, chemotaxonomy and phylogenetic analysis was used to characterise these isolates. A number of potentially novel strains belonging to the rarer genera were identified, including two KineococcusCape and three Micromonospora strains. Two novel Kribbella species were isolated from soil samples and the species descriptions of Kribbella karoonensis Q41 T and Kribbella swartbergensis HMC25 T were published inof 2006. More than 80% of all antibiotics are produced by actinomycetes and they are potentially the source of novel bioactive compounds. In this study, all actinomycete isolates were screened for antimicrobial activity against Mycobacterium aurum A+. Antibiotic production by the actinomycete strains was assessed by agar overlays, solvent extraction and bioautography, as well as by PCR screening with primers that target antibiotic biosynthetic genes. Twenty four actinomycete strains produced antimicrobial compounds that inhibited M. aurum A+. Antibiotic production was foundUniversity to be the most prevalent in strains isolated from soil, with Streptomyces being the most prolific antibiotic-producing genus. The antimycobacterial compound produced by Streptomyces speibonae strain PK-BlueT was characterised. Based on preliminary structural and physical characterisation, it appears that this compound is a tetramic acid type antibiotic. The 168 rRNA gene sequence similarity between members of the genus Kribbella ranges from 97.5% to 99.1%, and DNA-DNA hybridization is usually performed when classifying new species within this genus. The potential of the recN and gyrB genes to discriminate between Kribbella species was investigated. A plot of DNA relatedness versus pairwise gyrB sequence similarity was not linear. Kimura's 2-parameter model has been used in other actinomycete genera to determine the gyrB-based genetic distances and it is reported that a genetic distance of 0.014 can be used as the threshold for species delineation. In this study, the gyrB-based III genetic distance was determined between all Kribbella type strains and values were found to range from 0.0525 to 0.1495, confirming that the gyrB gene can distinguish between species of this genus. Based on the analysis of gyrB-based genetic distances versus DNA relatedness between Kribbella species, it is proposed that a gyrB based genetic distance of greater than 0.04 be set as the threshold for species delineation within the genus Kribbella. Phylogenetic analysis based on the gyrB gene was found to provide better resolution and more robust trees than those based on the 16S rRNA gene sequence. Based on this study, the gyrB gene can be used to resolve species relationships within the genus Kribbella. Town Cape of University IV Abbreviations A Adenine AFLP Amplified fragment length polymorphism AHBA 3-amino-5-hydroxy-benzoic acid AIDS Acquired immune deficiency syndrome ANI Average nucleotide identity ARDRA Amplified rONA restriction analysis BCG vaccine Bacille Calmette-Guerin vaccine Big Pharma Large pharmaceutical companies bp Base pair(s) C Cytosine CAS Cerium (IV) ammonium sulphate CDC Centers for Disease Control, USA CF Culture filtrate cm Centimetre(s) CZ agar Czapek solution agar Da Dalton(s) DAP 2,6-Diaminopimelic acid DOH DNA-DNA hybridization Town DGGE Denaturating gradient gel electrophoresis DNA Deoxyribonucleic acid dNTP deoxyribonucleoside triphosphate (dATP, dCTP, dGTP and dTTP) 001 2-deoxy-scyl/o-inosose DOTS Directly observed therapy, short-course DP Diffusible pigments Cape DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH 0 20 Deuterated water of EDTA Ethylenediaminetetra-acetic acid (disodium salt) ESMS Electrospray mass spectrometry FAME Fatty-acid methyl ester FDA Food and Drug Administration, USA FT-IR Fourier transform infrared spectroscopy g Gram(s) g Gravitational constant G GuanineUniversity G+Cmol% Mole percent guanine plus cytosine HBC High burden countries HIV Human immunodeficiency virus HM Hacene's Medium h Hour(s) HTS High throughput screening I Inosine ISP International Streptomyces Project ITS Internal transcribed spacer kb Kilobase(s) kDa Kilodaltons(s) km Kilometre( s) v I Litre(s) LFRFA Low-frequency restriction fragment analysis M Molar MA Modified Aretz medium (broth) MALDI-TOF Matrix adsorbed laser desorption/ionization time-of-flight mass spectrometry Mb Megabase(s) MC agar Modified Czapek solution agar MDR Multi-drug resistant mg Milligram(s) MIC Minimum inhibitory concentration min Minute(s) ml Millilitre(s) MLEE Multilocus enzyme electrophoresis MLSA Multilocus sequence analysis MLST Multilocus sequence typing mM Millimolar MRSA Methicillin-resistant Staphylococcus aureus MTB Mycobacterium tuberculosis MIT thiazolyl blue tetrazolium bromide MW Molecular weight mlz Mass-to-charge ratio ng Nanogram(s) Town NMR Nuclear magnetic resonance NRPS Non-ribosomal peptide synthetase nt Nucleotide(s) OD Optical density OD6oo Optical density at 600 nm Cape OTU Operational taxonomic unit PAGE Polyacrylamide gel electrophoresisof PBP Penicillin-binding proteins PCR Polymerase chain reaction PE agar Plant extract agar PFGE Pulse-field gel electrophoresis PKS Polyketide synthase PKS-I Type-I polyketide synthase PKS-II Type-II polyketide synthase PyMS Curie-point mass spectrometry R&D Research and development RAPD-PCR UniversityRandomly amplified polymorphic DNA-PCR RBR Relative binding ratio rDNA Ribosomal deoxyribonucleic acid RFLP Restriction fragment length polymorphism RIS-PCR Ribosomal intergenic spacer-PCR RNA Ribonucleic acid rRNA Ribosomal ribonucleic acid RT Room temperature (20-22°C) s Second(s) SC agar Starch-casein agar SEM Scanning electron microscopy SSC Standard short course chemotherapy Sub Substrate mycelium VI T Thymine TAE Tris-acetate-EDTA buffer TB Tuberculosis TE buffer 10 mM Tris-HCI, 1 mM EDTA, pH 7.8 Temp Temperature TGGE Temperature gradient gel electrophoresis TLC Thin layer chromatography Tm Melting temperature UK United Kingdom UPGMA Unweighted pair group method with arithmetic mean USA United States of America UV Ultraviolet V Volts Vol Volume v/v Volume per volume VRE Vancomycin-resistant enterococci w/v Weight per volume WHO World Health Organization XDR Extensively drug-resistant Town YEME Yeast extract-malt extract (ISP 2) medium z Area of the zone of inhibition in mm2 h Lambda DNA Cape I-Ig Microgram(s) I-Ig/ml Microgram(s) per millilitre of 1-11 Microlitre(s) % Percentage °C Degrees Celsius 1-0 One-dimensional 2-D Two-dimensional 7H9 Difco Middlebrook

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