Comparison of actinobacterial diversity in Marion Island terrestrial habitats Walter Tendai Sanyika A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biotechnology, University of the Western Cape. Supervisor: Prof D. A. Cowan November 2008 DECLARATION I declare that “Comparison of actinobacterial diversity in Marion Island terrestrial habitats” 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. Walter Tendai Sanyika November 2008 -------------------------------------- ii Abstract Marion Island is a sub-Antarctica Island that consists of well-characterised terrestrial habitats. A number of previous studies have been conducted, which involved the use of culture-dependent and culture-independent identification of bacterial communities from Antarctic and sub-Antarctic soils. Previous studies have shown that actinobacteria formed the majority of microorganisms frequently identified, suggesting that they were adapted to cold environments. In this study, metagenomic DNA was directly isolated from the soil and actinomycete, actinobacterial and bacterial 16S rRNA genes were amplified using specific primers. Hierarchical clustering and multidimensional scaling were used to relate the microbiological diversity to the habitat plant and soil physiochemical properties. The habitats clusters obtained were quite similar based on the analysis of soil and plant characteristics. However, the clusters obtained from the analysis of environmental factors were not similar to those obtained using microbiological diversity. The culture- independent studies were based on the 16S rRNA genes. Soil salinity was the major factor determining the distribution of microorganisms in habitats based on DGGE and PCA. Nutrient availability was also an important factor. The bacterial and actinobacterial diversities were mainly influenced by different combinations of soil and plant characteristics. The distribution of actinobacteria was mainly influenced by pH. In addition, total carbon, exchangeable magnesium and total potassium. The plants included Poa annua, mire bryophytes and the Bryum/ Breutelia plant species. The distribution of bacteria was influenced by all the forms of calcium and potassium, total sodium and exchangeable sodium. The plants included P. annua, lichens, and epiphytic graminoids and brachythecium mosses. The analysis of metagenomic clone libraries from three selected habitats suggested that oxygen availability was also an important determinant of microbiological diversity. Less than 1% of the phylotypes identified from the metagenomic libraries were identified as cultured. The rest showed 16S rRNA gene sequence identities of between 89 and 99% to other phylotypes. The actinobacteria identified belonged to the suborders, iii Acidimicrobineae, Propionibacterineae, Streptomycineae, Frankineae, Corynebacterineae, Streptosporangineae and Micrococcineae. Habitat MI 1.2 (Coastal Fellfield Habitat) was dominated by the Micrococcineae (44%) and Propionibacterineae (18%). This habitat had higher species richness, diversity indices and evenness compared to habitats MI 5.1 (Cotula Herbfield Habitat) and MI 6.3 (Wet Mire Habitat). Habitat MI 51 was dominated by the Frankineae (38%) and Streptosporangineae (32%) whilst MI 6.3 was dominated by the Acidimicrobineae (54%) and Frankineae (24%). These microorganisms dominating Habitats MI 5.1 and MI 6.3 were associated with mineralization activities and generation of energy from inorganic compounds under anaerobic conditions. This was attributed the presence of compacted peat soils in habitat MI 5.1 due to peats compacted by trampling from animals and water-logging in habitat MI 6.3. A total of 42 different actinomycetes were subsequently isolated using standard techniques and media. Only 1.3% of the actinomycete phylotypes identified from metagenomic clone libraries were subsequently isolated using culture-dependent techniques. The sequences from the rest of the isolates were not identified from metagenomic clone libraries, although were all related. The 13 isolates that showed less than 99% sequence identities to other known actinomycetes may be new species. Amongst these, 4 isolates showed sequence identities between 93 and 97% and may belong to new genera. This study has therefore, used both culture-dependent and culture-independent techniques to describe the community structures of three Marion Island terrestrial habitats. This resulted in the identification the major factors that determine the distribution of microorganisms, phylogenetic analysis of actinobacteria in three of the habitats and isolation of novel actinomycetes. The study showed that environmental characteristics are strong determinants that influence the spatial distribution of microorganisms on Marion Island and that some actinobacterial species that are endemic to the island. iv Dedication This thesis is dedicated to the man and woman who taught me how to read and write, and selflessly committed their lives to my education: My father, Mr Webster Evans Gwanzura, and my mother Mrs Joanita Zhakata (Gwanzura). v Acknowledgements I would like to thank the Lord God almighty for the guidance and looking over me through all the challenges during my studies. I would also like to thank the following people and organizations for providing me opportunities or support: 1. Professor Donald Cowan for giving me an opportunity to study for a PhD, for the knowledge, guidance, supervision, funding and support. I really appreciate all the support that you have given to me and my wife Fungi. 2. Dr William Stafford for the commitment to my work, selflessness, advice and knowledge. This work would not have been the same without your leadership. 3. Professor Mark Gibbons for assisting us with data analysis in Chapter 3. 4. The National Research Foundation (South Africa) for financing my studies. 5. Chodziwadziwa Kabudula for teaching me how to analyze my data and to use R. You were irreplaceable and your input laid the foundation of Chapter 3. 6. Dr Bernard Bulawayo for teaching me how to do research and looking over me all the time. 7. My wife Fungai for the love, patience and support. 8. Dr Heide Goodman for the ways in which you supported me and the administration of our daily requirements. 9. Moredreck Chibi for the friendship and social support. 10. Claudius Marondedze for the friendship and social support. 11. The Shofar Tygerberg Church for all the socio-spiritual support, especially to members from the cell group, Pastor Ross Van Niekerk and Pastor Theuns Pauw. 12. My family for the support and encouragement and all the prayers. 13. My friends for all the supportive roles that they have played in my life here in the diaspora. 14. The wonderful people from IMBM who have assisted me, taught me research skills and helped me find my way. A special thanks goes to Joseph D. W. Lako. vi Glossary and Abbreviations Abbreviation Definition APS Ammonium persulphate ARDRA Amplified Ribosomal DNA Restriction Analysis Bisacrylamide N, N-Methylene bisacrylamide BLAST Basic Local Alignment Search Tool CTAB Cetyl trimethyl ammonium bromide DGGE Denaturing Gradient gel electrophoresis DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid dNTP Deoxyribonucleotide triphosphate EDTA Ethylenediaminetetraacetic acid EtBr Ethidium bromide H Hour IPTG Isopropyl-beta-D-thiogalactopyranoside Min Minutes mM Millimolar Ng Nanogram PCA Principal Component Analysis PCR Polymerase Chain Reaction Pg Picogram PIPES Piperazine-1,2-bis[2-ethanesulfonic acid] PVPP Polyvinylpolypyrrolidone vii Table continued Abbreviation Definition Rcf Relative Centrifugal Force (xg) Rf Relative Retention Factor Rpm Revolutions per minute S Seconds SDS Sodium dodecyl Sulphate TAE Tris-acetate EDTA TBE Tris-borate EDTA TE Tris EDTA TEMED N,N,N',N'-Tetramethylethylenediamine X-gal 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside Μg Microgram Μl Microlitre viii Table of contents Content Page CHAPTER 1..........................................................................................................1 1.0 Literature review .............................................................................................1 1.1 Background and summary ..............................................................................1 1.1.1 Biodiversity at Marion Island.....................................................................3 1.1.2 Climate and climate change .....................................................................4 1.1.3 Energy production and nutrient recycling .................................................5 1.2 The Marion Island terrestrial habitats..............................................................6 1.2.1 The Coastal Salt-Spray Complex .............................................................8 1.2.2 The Fellfield Complex...............................................................................9 1.2.3 The Slope Complex..................................................................................9 1.2.4 The Biotic Grassland Complex ...............................................................11 1.2.5 The Biotic Herbfield Complex .................................................................12 1.2.6 The Mire Complex ..................................................................................13 1.2.7 The Polar Desert complex......................................................................14