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The Queuosine Pathway Is Not Essential for Ensifer Medicae WSM419 Symbiosis and Certain Components of This Pathway Modulate Lipid Biosynthesis

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The Queuosine Pathway Is Not Essential for Ensifer Medicae WSM419 Symbiosis and Certain Components of This Pathway Modulate Lipid Biosynthesis The Queuosine pathway is not essential for Ensifer medicae WSM419 symbiosis and certain components of this pathway modulate lipid biosynthesis By Jaco Daniel Zandberg Supervisors: Dr. Wayne Reeve Dr. Julie Ardley Dr. Ravi Tiwari i Declaration I hereby declare, that unless otherwise stated, the work presented in this thesis is my own Jaco Daniel Zandberg ii Acknowledgements I would like to begin with the formalities. A special thank you to Mr. Xin Du and Prof. Yonglin Ren at Murdoch University, Perth, Australia for allowing me access to your HS-GC system and analysis. Furthermore, thank you to Dr. Bill Dunstan for allowing me access to your microscope and imaging software. Now, with the formalities finished, I take the time here to use all the words I was barred from using by my supervisors whilst writing this thesis (because I’ll be damned if I don’t get to use these fantastic words) and what better place than here, thanking the very people that told me not to. So here I take the time to showcase my thanks to Dr. Julie “comma” Ardley for ensuring that I understand the plethora of information regarding everything ‘plant’ and for providing me with the necessary support when I needed it most. I thank Dr. Ravi “sticky fingers” Tiwari for “procuring” much needed reagents and for teaching me the most complicated genetic engineering technique, primer design. Lastly, I would like to thank Dr. Wayne “strikethrough” Reeve for providing me with a once in a lifetime opportunity, showcasing a phenomenal aptitude for the work done here and for excelling in his role as supervisor, one could not hope for a better supervisor. This one short year has been the most rewarded educational experience I have had in eighteen years of being educated, I sincerely thank again all those who were mentioned and all those who were not mentioned. iii Table of Contents DECLARATION .................................................................................................................................................. II ABBREVIATIONS ............................................................................................................................................. VII ABSTRACT ..................................................................................................................................................... VIII INTRODUCTION ................................................................................................................................................ 1 1.1 LIFE ON EARTH IS BASED ON A HIGHLY SIMILAR GENETIC CODE ................................................................. 2 1.2 THE FEATURES AND FUNCTIONALITIES OF THE GENOME............................................................................ 3 1.3 EXPRESSING THE GENETIC MESSAGE .......................................................................................................... 5 1.3.1 THE TRANSLATION MACHINERY AND THE UNIVERSAL GENETIC CODE ....................................................................... 5 1.3.2 DECODING THE GENETIC CODE .......................................................................................................................... 7 1.3.3 THE SELECTION-MUTATION DRIFT THEORY.......................................................................................................... 8 1.3.4 THE CODON-ANTICODON WOBBLE POSITION ....................................................................................................... 9 1.3.5 THE RIBOSOMAL GRIP AND ITS EFFECTS ON CODON-ANTICODON BINDING AFFINITY .................................................. 10 1.4 BROADENING THE WOBBLE-POSITION RULES .......................................................................................... 11 1.4.1 THE DEVIATION IN THE GENETIC CODE .............................................................................................................. 12 1.5 THE GROSJEAN & WESTHOF MODEL FOR THE UNIVERSAL GENETIC CODE ................................................ 12 1.6 THE GLOBAL AND INDUCIBLE MODULATION OF THE TRANSLATION MACHINERY .................................... 14 1.7 TRNA MODIFICATION ............................................................................................................................... 15 1.8 THE QUEUOSINE TRNA MODIFICATION .................................................................................................... 18 1.9 CONTROLLING THE QUEUOSINE PATHWAY .............................................................................................. 23 1.10 THE IMPACT AND FUNCTION OF QUEUOSINE ......................................................................................... 26 1.11 LEGUMES AND ROOT-NODULE BACTERIA ............................................................................................... 29 1.11.1 ESTABLISHMENT OF AN AMICABLE SYMBIOTIC RELATIONSHIP .............................................................................. 29 1.11.1.1 The rhizosphere ............................................................................................................................. 29 1.11.1.2 The infection process of RNB ......................................................................................................... 31 1.11.2 ESTABLISHMENT OF AN N2 FIXING SYMBIONT .................................................................................................. 33 1.11.3 AN EXAMPLE OF TERMINALLY DIFFERENTIATED BACTEROIDS ............................................................................... 34 1.12 WHAT IS THE ROLE OF THE Q-PATHWAY IN RNB? ................................................................................... 35 1.14 AIMS ....................................................................................................................................................... 36 MATERIALS AND METHODS ........................................................................................................................... 37 2.1 BIOINFORMATICAL ANALYSIS AND PREDICTION ....................................................................................... 38 2.1.1 ASSEMBLING GENETIC SEQUENCES OF CONSTRUCTS ............................................................................................ 39 2.2 STRAINS, GROWTH CONDITIONS AND CRYOPRESERVATION .................................................................... 40 2.3 PCR PRIMER DESIGN ................................................................................................................................. 43 2.4 PCR AMPLIFICATION CONDITIONS ............................................................................................................ 44 2.5 PCR AMPLIFICATION ................................................................................................................................. 45 2.6 PCR PRODUCT PURIFICATION ................................................................................................................... 46 iv 2.7 LIGATION REACTIONS ............................................................................................................................... 46 2.7.1 QUADRUPLE FRAGMENT LIGATION CONSTRUCTS PQLΔSMED_3534, PQLΔSMED_4938 AND PQLΔSMED_4937 IN THE MOBILE SUICIDE VECTOR PJQ200SK. ....................................................................................................................... 46 2.7.1 QUADRUPLE FRAGMENT LIGATION CONSTRUCTS PQLΔNGR_C36640 IN THE MOBILE SUICIDE VECTOR PJQ200SK. ..... 46 2.8 COMPETENT CELL PREPARATION .............................................................................................................. 47 2.9 EXTRACTION AND PURIFICATION OF PLASMID DNA ................................................................................. 48 2.10 TRANSFORMATION ................................................................................................................................ 48 2.11 BIPARENTAL CONJUGATION METHODOLOGY WITH E. MEDICAE WSM419 ............................................. 49 2.12 TRIPARENTAL CONJUGATION METHODOLOGY WITH E. FREDII NGR234 ................................................. 50 2.13 PCR CONFIRMATION............................................................................................................................... 51 2.13.1 PCR AMPLIFICATION CONFIRMATION OF THE UP AND DN REGIONS FOR INACTIVATION VECTORS ............................. 51 2.13.2 PCR AMPLIFICATION CONFIRMATION OF THE UP AND DN REGIONS FOR MUTANT DERIVATIVES ............................... 51 2.14 AGAROSE GEL ELECTROPHORESIS ........................................................................................................... 52 2.15 DNA SEQUENCING .................................................................................................................................. 53 2.16 ANTIBIOTIC VIABILITY TESTING ............................................................................................................... 53 2.17 MEAN GENERATION TIME ASSAY ........................................................................................................... 53 2.18 PROLONGED STATIONARY PHASE ASSAY ................................................................................................ 54 2.19 E. MEDICAE WSM419 AND MUTANT DERIVATIVE STRESS PHENOTYPING............................................... 55 2.19.1 MOTILITY PHENOTYPING .............................................................................................................................
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