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4.3.4 Phylogenetic and Sequence Analysis

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4.3.4 Phylogenetic and Sequence Analysis Town The copyright of this thesis rests with the University of Cape Town. No quotation from it or information derivedCape from it is to be published without full acknowledgement of theof source. The thesis is to be used for private study or non-commercial research purposes only. University SELECTIVE ISOLATION AND CHARACTERISATION OF INDIGENOUS ACTINOBACTERIA, WITH PARTICULAR EMPHASIS ON THE GENUS Amycolatopsis Town by Cape of Gareth John Everest University Thesis presented for the Degree of Doctor of Philosophy in the Department of Molecular and Cell Biology, Faculty of Science, University of Cape Town, South Africa. February 2010 2 Town Cape of University 3 Table of Contents Acknowledgments 5 List of Abbreviations 6 Abstract 10 Chapter 1 13 Introduction Chapter 2 89 Actinobacterial isolation and preliminary identification, antibiotic screening and extraction Town Chapter 3 123 Identification and characterisation of isolated actinobacteria Chapter 4 Cape 175 The use of gyrB and recN gene sequences in the phylogenetic analysis of the genus Amycolatopsis of Chapter 5 213 General discussion Appendices 221 University 4 Town Cape of University 5 Acknowledgements First and foremost I would like to thank my supervisor Dr Paul Meyers for his continued support, guidance and encouragement throughout this project. His enthusiasm towards research is contagious and has most certainly rubbed off during the five years under his supervision, something for which I will always be in his debt. I am also grateful to the National Research Foundation and the University Scholarships Committee (UCT) for financial support throughout my studies, without which it would have been difficult for me to have reached this point. Town My thanks must also go out to all those who in some way or another contributed to the work in this thesis: to Di James and Bruna Galvão for DNA sequencing; Miranda Waldron for her assistance with scanning electron microscopy; Jerome Diedericks forCape collecting the soil sample from which I performed the isolation and Professor J. P. Euzébyof and Professor Dr H. G. Trüper for assistance with deriving the species names for four of my strains. Further thanks must be said to all my past and present lab mates – Andrew, Bronwyn, Darren, Henrique, Iulia, Jeff, Marilize,University Saeed and all the Honours students who have passed through the lab, for all their help with experiments, opinions and interpretation of results and for making the time spent in the lab mostly fun and always memorable. Last, but certainly by no means least, a big thank you to my mother, father and brother for all their love and support throughout my studies at UCT and for putting up with me over the years, I’m sure it wasn’t always easy. 6 List of Abbreviations 7H9 - Middlebrook 7H9 (agar/broth) A - adenine (DNA base) ACD - albumin-dextrose-catalase ADRA - amplified DNA restriction analysis AFLP - amplified fragment length polymorphism ANI - average nucleotide identity ARDRA - amplified ribosomal DNA restriction analysis Ala - alanine A-site - aminoacyl site on the ribosome ATCC - American Type Culture Collection ATP - adenosine triphosphate Town bp - base pairs (DNA) BLAST - basic local alignment search tool blastn - nucleotide BLAST Cape C - cytosine (DNA base) CAS - cerium (IV) ammoniumof sulphate cm - centimeters CZ - Czapek solution (agar/broth) DAP - diaminopimelic acid DDH - DNA-DNA hybridisation DGGE - denaturing gradient gel electrophoresis DMSO - Universitydimethyl sulfoxide DNA - deoxyribonucleic acid DOI - 2-deoxy scyllo inosose DOTS - directly observed treatment, short course DPG - diphosphatidylglycerol DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen dNTP - deoxyribonucleotide triphosphate EDTA - ethylenediamine tetraacetic acid e.g. - exempli gratia, “for the sake of example” (for example) 7 etc - et cetera, “and the rest” or “so forth” Fig - figure fMET - formylmethionine (proteinogenic amino acid) g - grams G - guanine (DNA base) GBDP - genomic BLAST distance phylogeny gly - glycerol h - hours HGT - horizontal gene transfer HIV - human immunodeficiency virus HPLC - high performance liquid chromatography I - inosine (nucleoside) i.e. - id est, “it is” or “that is (to say)” ISP - International Streptomyces Project Town ITS - internally transcribed spacer I.U. - International Unit kb - kilobase pairs (103 bp) (DNA)Cape km - kilometers of km2 - square kilometers KRCA - Kenilworth Racecourse conservation area KZN - Kwa-Zulu Natal Province l - liter LB - Luria-Bertani (agar/broth) m - Universitymeters M - molar MALDI-TOF - matrix-assisted laser desorption/ionization – time-of-flight Mb - megabase pairs (106 bp) (DNA) MC - modified Czapek solution (agar/broth) MDR - multidrug-resistant mg - milligrams min - minutes ml - milliliters MLSA - multilocus sequence analysis 8 mm - millimeters mm2 - square millimeters mM - millimolar MRSA - methicillin-resistant Staphylococcus aureus MTT - thiazolyl blue tetrazolium bromide ng - nanograms nm - nanometers nt - nucleotides (DNA) OD - optical density PBP - penicillin binding proteins PC - phosphatidylcholine PCR - polymerase chain reaction PE - phosphatidylethanolamine PG - phosphatidylglycerol Town PI - phosphatidylinositol PIMs - phosphatidylinositol mannosides PME - phosphatidylmethylethanolamineCape RAPD - randomly amplified ofpolymorphic DNA Rf - retention factor RFLP - restriction fragment length polymorphism RNA - ribonucleic acid mRNA - messenger RNA rRNA - ribosomal RNA tRNA - Universitytransfer RNA rpm - revolutions per minute s - seconds Sac. - Saccharopolyspora Sal. - Salinispora SCFA - short chain fatty acids SE - soil extract (agar) SEM - scanning electron microscopy Ser - serine sp. - species (singular) 9 sp. nov. - species nova, “new species” spp. - species (plural) Sta. - Streptoalloteichus subsp. - subspecies T - thymine (DNA base) TA - annealing temperature TAE - tris-acetate EDTA buffer TE - tris-HCl/EDTA buffer TB - tuberculosis TLC - thin layer chromatography Tm - melting temperature U - units UV - ultraviolet V - volts Town vs - versus v/v - volume for volume WHO - World Health OrganizationCape w/v - weight for volume of XDR - extensively drug-resistant x g - times gravity (g-force – relative centrifugal force) YEME - yeast extract malt extract (agar/broth) °C - degrees Celsius > - “greater than” or “more than” ≥ - University“greater than or equal to” µg - micrograms µl - microliters µM - micromolar λ - phage Lambda 10 Abstract A soil sample collected from within the fynbos-rich area that is surrounded by the horseracing track at Kenilworth Racecourse, Cape Town, served as the source for the isolation of filamentous actinobacteria. The sampling area is known to contain a wide range of biodiversity, including endemic and endangered plant species. A total of 112 bacterial strains were initially isolated and, following morphological examination and de-replication, 64 strains were presumptively identified as filamentous actinobacteria and screened for their ability to produce antibiotics active against Mycobacterium aurum A+, a non-pathogenic, fast growing mycobacterium with a similar antibiotic susceptibility profile to that of Mycobacterium tuberculosis. Moderate to very strong antimycobacterial activity was recorded for 31 isolates and all were identified to belong to the genus Streptomyces, based on a rapid identification method. Based solely on morphological examination, a further 17 isolates were noted as interesting and selected for preliminaryTown identification as well. Eight of these morphologically interesting isolates were identified to belong to the genus Streptomyces, with three being identified as Amycolatopsis, three as belonging to members of the family Micromonosporaceae, one to the genus Nocardia, oneCape to Gordonia , Nocardia or Skermania and one to either Kribbella or Nocardioides. The nine isolates with the highest antimycobacterial activity were further screened for activity against Escherichiaof coli and Staphylococcus aureus, subjected to antibiotic extraction and attempts were made to partially purify the active compounds. Only a weakly active compound from one of the Streptomyces strains was successfully isolated by column chromatography. The genera to which the top nine antibiotic producing strains belong, as well as the genera to which the strains identified as Universitynon-Streptomyces by the rapid molecular identification method belong, were definitively determined by BLAST analysis of their 16S rRNA gene sequences. The closest relatives were determined by 16S rRNA gene and gyrB gene based phylogenetic analyses. All strains were subjected to physiological characterisation to allow them to be differentiated from the most closely related type strains with validly-published names. Three strains belonging to the genus Amycolatopsis were shown to be distinct from all closely related type species by gyrB sequence analysis, with DNA-DNA hybridisation and physiological differences confirming this. The single Kribbella isolate was shown to be distinct by DNA-DNA hybridisation. Two strains belonging to the genus Micromonospora showed a high level of similarity to each other and could not be 11 differentiated. However, they showed a high number of physiological differences to the closely related type strain of Micromonospora olivasterospora and are likely to be distinct from this species. Two isolated Nocardia strains seem likely to represent novel species, showing multiple physiological differences from their respective relatives (‘Nocardia rhamnosiphila’,
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