Biodiscovery and Biodiversity of Antarctic Bacteria by Jimmy Twin B.App.Sci. (Queensland University of Technology, Australia) B.Sc. Hons (University of Queensland, Australia) Submitted in fulfilment of The requirements for the degree of Doctor of Philosophy University of Tasmania Hobart Tasmania Australia July 2008 UTAS Prologue Declaration This thesis contains no material that has been accepted for the award of any other degree or diploma in any te1tiary institution. To the best of my knowledge, this thesis does not contain material written or published by another person, except where due reference is made Jimmy Twin University of Tasmania Hobart July 2008 This thesis may be made available for loan and limited copying in accordance with the Copyright Act 1968 II Prologue Contents LIST OF TABLES ............................................................................................................................... IV LIST OF FIGURES .............................................................................................................................. V ABBREVIATIONS .............................................................................................................................. VI ABSTRACT VII ACKNOWLEDGEMENTS ................................................................................................................. IX PUBLICATIONS AND PRESENTATIONS RESULTING FROM THIS THESIS ...................... XI CHAPTER 1 OVERVIEW ................................................................................................................... 1 1.1 INTRODUCTION .......................................................................................................................... 2 1.2 ANTARCTIC MICROBIOLOGY ...................................................................................................... 3 1.3 AIMS OF THIS PROJECT ................................................................................................................ 6 CHAPTER 2 TAXONOMY OF ANTARCTIC ACTINOBACTERIA ISOLATES .......................... 8 2.1 INTRODUCTION ........................................................................................................................... 9 2.2 MATERIALS AND METHODS ....................................................................................................... 15 2.3 RESULTS AND DISCUSSION ....................................................................................................... 22 CHAPTER 3 THE UTILISATION OF HYDROCARBONS BY ANTARCTIC ACTINOBACTERIA ............................................................................................................................. 35 3.1 INTRODUCTION ......................................................................................................................... 36 3 .2 MATERIALS AND METHODS ....................................................................................................... 41 3.3 RESULTS ................................................................................................................................... 45 3.4 DISCUSSION .............................................................................................................................. 51 CHAPTER 4 ANTIMICROBIAL SCREENING OF ANTARCTIC ACTINOBACTERIA .......... 54 4.1 INTRODUCTION ....................................................................................................................... 55 4.2 MATERIALS AND METHODS ....................................................................................................... 59 4.3 RESULTS ................................................................................................................................... 61 4.4 DISCUSSION .............................................................................................................................. 67 CHAPTER 5 BACTERIAL DIVERSITY OF EPIGLACIAL LAKES OF THE FRAMNES MOUNTAINS ....................................................................................................................................... 71 5.1 INTRODUCTION ..................................................................................................................... 72 5.2 MATERIALS AND METHODS ...................................................................................................... 84 5.3 RESULTS .................................................................................................................................. 89 5.4 DISCUSSION .............................................................................................................................. 94 CHAPTER 6 ANALYSIS OF BACTERIA TRAPPED WITHIN ANTARCTIC ICE .................. 98 6.1 INTRODUCTION ....................................................................................................................... 99 6.2 MATERIALS AND METHODS ................................................................................................... 105 6.3 RESULTS ................................................................................................................................ 109 6.4 DISCUSSION .......................................................................................................................... 114 CHAPTER 7 GENERAL DISCUSSION ......................................................................................... 118 7.1 OUTCOMES OF THIS STUDY .................................................................................................... 118 7.2 FUTUREDIRECTIONS ............................................................................................................... 119 CHAPTER 8 REFERENCES ........................................................................................................... 120 111 Prologue List of Tables Table 2a A comparison of biochemical and carbon utilisation characteristics of UTAA strains compared to their closest 168 rRNA neighbour. Page 26. Table 3a Growth ofUTAA isolates in the presence of various alkanes. Page 46. Table 3b Growth of UTAA isolates in the presence of various P AHs. Page 4 7. Table 4a Summary of antimicrobial screening of known producing UTAA isolates against Listeria monocytogenes strains. Page 61. Table 4b Summary of antimicrobial screening of known producing UTAA isolates against Listeria monocytogenes strains. Page 62. Table 4c Summary of antimicrobial screening of select UTAA isolates against various bacterial strains. Page 66. Table 4d Pigmentation ofUTAA1477 and Steptomyces sanglieri. Page 66. Table 5a Physicochemical characteristics oflake samples obtained for this study. Page 78. Table 6a BLAST matches of CAIS399 16S rRNA gene clones. Page 110. Table 6b BLAST matches of Law Dome 16S rRNA gene clones. Page 113. lV Prologue List of Figures Figure 2i 16S rRNA gene Neighbour Joining tree ofUTAA isolates with closest relatives. Page 24. Figure 2ii SEM micrograph of stationary Promicromonospora UTAA143 cells. Page 31. Figure 3i Neighbour Joining tree of alkane degradation gene sequences. Page 49. Figure 3ii Neighbour Joining tree of naphthalene degradation gene sequences. Page 50. Figure 4i 16S rRNA gene Neighbour Joining tree of Streptomyces isolates. Page 64. Figure 5i Location ofFramnes Mountains Lakes (Pickard, 1982). Page 74. Figure 5ii Plot showing the pH of the water along the entire depth of each epiglacial lake in this study. Page 77. Figure 5iii 16S rRNA gene Neighbour Joining tree of Patterned Lake clones with closest matches obtained from Genbank. Page 91. Figure 5iv 16S rRNA gene Neighbour Joining tree of Sonic-Top clones with closest cultured and clone matches from Genbank. Page 92. Figure 5v t-RFLP analysis of 16S rRNA gene sequences obtained from lake filter samples from epiglacial lakes in the Framnes Mountains. Page 93. Figure 6i Map of East Antarctica. Page 103. Figure 6ii t-RFLP analysis of 16S rRNA gene sequences obtained from CAIS ice melt samples. Page 111. v Prologue The following abbreviations and acronyms have been used in this thesis: 16S rRNA gene 16 subunit ribosomal ribonucleic acid ACECRC Antarctic Climate and Ecosystem Cooperative Research Centre alk Alkane monooxygenase gene BH Bushnell Haas Cat.# Catalogue number CFB Cytophaga-Flavobacterium-Bacteriodes phylum dH20 Distilled water DNA Deoxyribonucleic acid DSMZ Deutsche sammlung von mikroorganismen und zellkulturen (German collection of micro organisms and cell cultures) G+C Guanine plus Cytosine GYM Glucose yeast malt media JCM Japan collection of micro organisms Kyr Thousand year( s) MA MarineAgar MDS Multi-dimensional scaling MK Menaquinone No. Number ndo Naphthalene dioxygenase gene OA Oatmeal Agar PCR Polymerase Chain Reaction PKS-II Polyketide synthase type II Pty ltd Proprietary limited RT Room temperature SAB Special Antarctic blend diesel SEM Scanning electron microscope Spp. Species t-RFLP Terminal restriction fragment length polymorphism TSA Tryptic soy agar TSA-ye Tryptic soy agar supplemented with yeast extract TSB Tryptic soy broth UTAA University of Tasmania Antarctic Actinobacteria (collection) Vl Prologue This PhD dissertation explores aspects of the microbial ecology of Antarctica, focussing on the taxonomy and physiological attributes of Antarctic bacteria, as well as investigating the bacterial diversity of previously uncharacterised Antarctic environments. The University of Tasmania has in its possession a collection of 1600 Actinobacteria strains previously isolated from Antarctic and sub-Antarctic soils. A polyphasic study