ACTINOBACTERIAL DIVERSITY OF THE ETHIOPIAN RIFT VALLEY LAKES By Gerda Du Plessis Submitted in partial fulfillment of the requirements for the degree of Magister Scientiae (M.Sc.) in the Department of Biotechnology, University of the Western Cape Supervisor: Prof. D.A. Cowan Co-Supervisor: Dr. I.M. Tuffin November 2011 DECLARATION I declare that „The Actinobacterial diversity of the Ethiopian Rift Valley Lakes is my own work, that it has not been submitted for any degree or examination in any other university, and that all the sources I have used or quoted have been indicated and acknowledged by complete references. ------------------------------------------------- Gerda Du Plessis ii ABSTRACT The class Actinobacteria consists of a heterogeneous group of filamentous, Gram-positive bacteria that colonise most terrestrial and aquatic environments. The industrial and biotechnological importance of the secondary metabolites produced by members of this class has propelled it into the forefront of metagenomic studies. The Ethiopian Rift Valley lakes are characterized by several physical extremes, making it a polyextremophilic environment and a possible untapped source of novel actinobacterial species. The aims of the current study were to identify and compare the eubacterial diversity between three geographically divided soda lakes within the ERV focusing on the actinobacterial subpopulation. This was done by means of a culture-dependent (classical culturing) and culture-independent (DGGE and ARDRA) approach. The results indicate that the eubacterial 16S rRNA gene libraries were similar in composition with a predominance of α-Proteobacteria and Firmicutes in all three lakes. Conversely, the actinobacterial 16S rRNA gene libraries were significantly different and could be used to distinguish between sites. The actinobacterial OTUs detected belonged to both the Rubrobacterales and Actinomycetales orders with members of the genus Arthrobacter being found in all three lakes. Geochemical properties were significantly different between the lakes, although more than one property attributed to the variance between community compositions. The diversity detected in the culture-based study differed significantly and all isolates belonged to the genus Streptomyces. Two novel strains were characterized by means of phylogenetic (16S rRNA gene sequence), physiological, morphological and biochemical analyses. Both novel isolates were capable of growing under “extreme” conditions- pH 12, 10% NaCl and 45°C. Partial enzyme characterization revealed that both strains produced xylanase enzymes that were active at pH 6.5 and 8.5 with an increase in activity up to 45°C. The results obtained revealed a previously undetected diversity of actinobacteria in the Ethiopian Rift Valley with a potentially novel subpopulation adapted to haloalkaline conditions. The low 16S rRNA sequence similarity of a substantial proportion of the libraries suggests that culture-based isolation may play a vital role in deciphering the community fingerprint. iii PUBLICATIONS ORIGINATING FROM THIS THESIS Poster/Presentations 1. Soil Metagenomics Symposium 2010. Berlin: Germany. Poster 2. Biotechnology Departmental open day 2011. Cape Town. Poster 3. South African Society for Microbiology conference 2011. Cape Town. Poster iv ACKNOWLEDGEMENTS I would like to extend my gratitude to: v My supervisors for their effort and time. v Dr. Bronwyn Kirby, for her unfailing guidance and motivation when I needed it the most. v Dr. Lise Øvreås and everyone at UIB for the opportunity to present my work. v Everyone at IMBM for their support and motivation, especially Dr. Heidi Goodman for the endless hours of work. v My family, especially Johan because without his patience and undying love I would have faltered long ago. v The National Research Foundation and the Norwegian research counsel for funding allocated to the project. v DEDICATION This thesis is dedicated to Johan C. Theron and Roelof Du Plessis vi CONTENTS DECLARATION ........................................................................................................................................................ II ABSTRACT ……………………………………………………………………………………………………………………………………………………….III PUBLICATIONS ORIGINATING FROM THIS THESIS .................................................................................................. IV ACKNOWLEDGEMENTS .......................................................................................................................................... V DEDICATION ......................................................................................................................................................... VI LIST OF TABLES ....................................................................................................................................................... X LIST OF EQUATIONS ............................................................................................................................................ XIII LIST OF ABBREVIATIONS.………………………………………………………………………………………………………………………………… XIV CHAPTER 1: LITERATURE REVIEW ........................................................................................................................... 1 1.1 INTRODUCTION TO HALOALKALINE ENVIRONMENTS ........................................................................................................ 1 1.2 SODA LAKE ENVIRONMENT ........................................................................................................................................ 2 1.2.1 Genesis of Soda lakes ................................................................................................................................ 2 1.2.2 The Ethiopian Rift Valley (ERV) .................................................................................................................. 4 1.3 MICROBIAL ECOLOGY OF SODA LAKES/ALKALINE BIODIVERSITY .......................................................................................... 5 1.3.1 Eubacterial placement within a wider ecological niche............................................................................. 5 1.3.2. Phototrophic primary producers .............................................................................................................. 7 1.3.3. Carbon cycle ............................................................................................................................................. 7 1.3.4. Sulphur cycle ........................................................................................................................................... 11 1.3.5. Nitrogen cycle ......................................................................................................................................... 11 1.4. ACTINOBACTERIA ................................................................................................................................................. 12 1.5. THEORETICAL ADAPTIVE MECHANISMS: CELLULAR EVOLUTION ....................................................................................... 15 1.6 RIBOSOMAL RNA GENES AND MOLECULAR MICROBIAL ECOLOGY .................................................................................... 16 1.6.1 The role of taxonomy in phylogenetic studies ......................................................................................... 16 1.6.2. A polyphasic approach towards actinobacterial taxonomy ................................................................... 19 1.6.3. Informational macromolecules: 16S rRNA as a priori............................................................................ 22 1.7. CULTURE VS CULTURE-INDEPENDENT ........................................................................................................................ 24 1.7.1. Limitations and advantages of phenetic or culture based classification ................................................ 25 1.7.2. Culture-independent approaches to phylogenetic diversity ................................................................... 26 1.8. BIOTECHNOLOGICAL AND INDUSTRIAL APPLICATION ..................................................................................................... 30 CHAPTER 2: MATERIALS AND METHODS .............................................................................................................. 34 2.1. ENVIRONMENTAL SAMPLE COLLECTION ..................................................................................................................... 34 2.2. DNA EXTRACTIONS .............................................................................................................................................. 35 2.2.1. Extraction of DNA from sediment samples ............................................................................................. 35 2.2.2. Preparation of genomic DNA from pure bacterial cultures .................................................................... 35 vii 2.2.4. Determination of DNA concentration ..................................................................................................... 35 2.3. POLYMERASE CHAIN REACTION (PCR) ...................................................................................................................... 36 2.3.1. Oligonucleotide primers and protocols .................................................................................................. 36 2.3.2. Purification of PCR products ..................................................................................................................