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Isolation and Characterization of Mutants Affecting Carbon Catabolism in Sín O Rh I Zob Ium Melilo Ti

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Isolation and Characterization of Mutants Affecting Carbon Catabolism in Sín O Rh I Zob Ium Melilo Ti Isolation and characterization of mutants affecting carbon catabolism in Sín o rh i zob ium melilo ti by Nathan lohn Poysti A Thesis submitted to the Faculty of Graduate studies of the university of Manitoba in partial fulfilment of the requirements of the degree of Master of Science Department of Microbiology University of Manitoba Winnipeg, Manitoba Canada @ Nathan Poysti ,2002 THE UNTVERSITY OF MANITOBA FACULTY OF GRADUATE STTIDIES tr*rrtr* COPYRIGHT PERMISSION lsolation and characterization of mutants Affecting carbon catabolism in Sinorhizobium meliloti BY Nathan John Poysti A ThesislPracticum submitted to the Faculty of Graduate Studies of The University of Manitoba in partial fulfillment of the requirement of the degree MASTER OF SCIENCE Nathan John Poysti O 2007 Permission has been granted to the University of Manitoba Libraries to lend a copy of this thesis/practicum, to Library and Archives Canada (LAC) to lend a copy of this thesis/practicum, and to LAC's agent (UMlÆroQuest) to microfilm, sell copies and to pubtish an abstract of this thesis/practicum. This reproduction or copy of this thesis has been made availabte by authority of the copyright owner solely for the purpose of private study and research, and may only be reproduced and copied as permitted by copyright laws or with express written authorization from the copyright owner. Abstract Sinorhizobium meliloti is a bacterium capable of participating in a symbiotic relation- ship with plants in which it forms N2 fixing nodules. The ability to use specific carbon sources has been shotnm to provide S. melíIoti and other rhizobia with competitive ad- vantages in the ability to effectively nodulate host plants. The sugars rhamnose and ara- binose were hypothesized to play a role in the abitity to compete for nodule occupancy in S. melilori. To test these hypotheses, the S. meliloti wild type, RmI02l, was subjected to transposon mutagenesis experiments and the resulting mutants were screened for the inability to use rhamnose or arabinose as sole carbon sources. Mutations affecting rhamnose and arabinose catabolism were isolated and subsequent characterization re- sulted in the identification of a rhamnose catabolism operon, an arabinose catabolism operon and a triose phosphate isomerase encoding gene. A-lthough mutants unable to utilize arabinose were not less competitive than wild type, the ability to utilize rhamnose was found to play a role in competition for nodule occupancy. Furthermore, the roles of two triose phosphate isomerase genes were characterized with respect to symbiosis and the free-living state. II Acknowledgements First, I would like to sincerely thank my supervisor, Dr. Ivan Oresnik, for his motivation, enthusiasm and support during this project. I am very glad to have found such a ded- icated supervisor! I would also like to thank my advisory committee: Dr. Harry Duck- worth and Dr. Teri de Kievit, for their thorough consideration and excellent suggestions regarding my work and thesis. i would like to thank my friends in the lab: Iason Richardson, Mark Miller_Williams, Brad Pickering, Erin Loewen, and HarryYudistira. Not only did we have excellent scien- tific discussions, but we had a lot of fun too! I am very grateful to both my immediate and extended family for being so enthusi- astic and encouraging during my studies. Last, but not least, Kimberly Poysti has been incredible. I sincerely appreciate her dedication, love, and patience. I would like to acknowledge the Natural Sciences and Engineering Research Council of Canada fo¡ their generous funding. III Contents Abstract II Acknowledgements IiI List of tables. VII List of figures . VIII I-ist of abbreviations x Introduction I S. nteliloti symbiosis and nodulation Ja S. meliloti carbon metabolism 7 Bacteroid metabolism 2l Competition for nodule occupancy . 25 ca Research goals J¿ Rhamnose catabolism and transport in S. melílotí 34 Summary 34 Introduction . 35 Materials and methods . 36 Bacterial strains, plasmids, and media 36 Genetic techniques 3B Rhamnose transport assays 3B Plantassays .... 3B Results 39 S. meliloti rhamnose catabolism Iocus 39 Rhamnose mutants are not sensitive on rhamnose/glycerol media 40 Heterologous complementation of S. meliloti rhamnose mutants 43 R. leguminosa.rum RhaK is capable of complementing S. meliloti rhaK mutations. 46 Testing the S. meliloti rhalocus for uptake of rhamnose. 47 s. meliloti rhamnose mutants are less competitive for nodule occupancy 50 Discussion 52 ry Characterization of S. m.eliloti triose phosphate isomerase genes 57 Summary 57 Introduction 5B Materials and methods . 59 Bacterial strains, plasmids, and media 59 DNA manipulations and plasmid constructions . 60 Genetic techniques and mutant construction. 63 Mutant identification . 64 liiose phosphate isomerase assays 65 Symbiotic competence assays 65 Results 66 Analysis of tpiAand tpiB 66 tpiAand tpiB are needed for gluconeogenesis 69 Both tpíAand tpiB encode triose phosphate isomerases 69 In uiuo complementation of tpiA 72 Constitutively expressed tpiA cannot complement tpiB . /J S)¡mbiotic competence phenotypes . 75 Discussion 79 Arabinose catabolism and transport in S. meliloti B4 Summary B4 Introduction B5 Methods UIoa Bacterial strains and growth conditions 87 Genetic techniques B9 Mutant identification. B9 Arabinose dehydrogenase assay. 90 B - galactosidase assays 9l Arabinose and glucose transport assays. 9l Symbiotic profi ciency assays. 92 Symbiotic competition assays. 92 Iìesults 93 Isolation of arabinose catabolism mutants. 93 Analysis of arabinose transcriptional unit. 94 Arabinose dehydrogenase activity is present in an araD mutant. 99 Induction of araoperon by arabinose, seed exudate and galactose. r00 Expression of the ara opeÍon is moderately repressed by succinate and glucose. I02 The araoperon is required for arabinose transport. 104 Glucose uptake is not compromised in arabinose catabolism mutants. 106 V Plant symbiosis and competition for nodule occupancy. r07 Discussion 108 Conclusion tl3 References tt7 VI List of tables 2.1 Bacterial strains and plasmids . J/ 2.2 Homology of S. meliloti and R.leguminosa.rumrhamnose loci 42 1) L.J Growth of s. melilofi rhamnose mutants on rhamnose and glycerol 44 2.4 The A. leguminosarum rharegion does not complement s. meliloti 45 2.5 R. leguminosarum rhaK complements s. melitoti rhaK mutations . 48 3.1 Bacterial strains and plasmids . 6i 3.2 Primers used in this work 62 3.3 Relevant growth phenotypes of triose phosphate isomerase mutants on defined media 70 3.4 Triose phosphate isomerase activities measured for different strains . ZI 3.5 complementation of triose phosphate isomerase mutants by plasmid ex- pressed tpiAandtpiB . Z4 3.6 Dry weights from growth trials of various S. metiloti strains Z6 4.1 Bacterial strains and plasmids . BB 4.2 Induction of the arabinose operon by several carbon sources l0I 4.3 The arabinose operon is not subject to catabolite repression by glucose or succinate 103 4.4 Labeled arabinose accumulation bywild tlpe and arabinose mutants . 105 VII Líst of figures o 1.1 Schematic of S. melilori hexose metabolism O L.2 S. meliloti galactose catabolism via the De Ley-Doudoroff pathway . i5 1.3 Schematic of the pentose phosphate pathway I7 I.4 Schematic of the S. meliloti tricarboxylic acid cycle . I9 2.I S. meliloti rhamnose utilization locus . 4I 2.2 s. meliloti wild type and mutant accumulation of labeled rhamnose . 49 2.3 Rhamnose accumulation by s. melilotí grown in rhamnose and glycerol . 51 2.4 A rhamnose mutant is less competitive than wild type for nodule occu- pancy 3.t Physical map of S. meliloti triose phosphate isomerase encoding loci . 3.2 Nodulation kinetics of S. nteliloti wild type and triose phosphate isomer- ase mutants 4.r Physical map of S. meliloti arabinose utilization locus . 95 4.2 Schematic of the S. melilotí arabinose catabolism pathway 9B 4.3 Á,n arabinose catabolism mutant is not competitively deficient 109 VIII List of abbreviations ABC ATP-binding cassette ATP Adenosine triphosphate CS Citrate slmthase DHAP Dihydroxyacetonephosphate DME Diphosphopyridine-nucleotidedependentmalicenzyme DNA Deoxyribonucleic acid ED Entner-Doudoroff EMP Embden-Meyerhof-Parnas EPS Exopolysaccharide FBA Fructose-bisphosphate aldolase G3P Glyceraldehyde-3-phosphate GAP Glyceraldehyde-3-phosphate dehydrogenase GC-MS Gaschromatography-massspectrometry GDH Glucose dehydrogenase Gm Gentamicin ICD Isocitrate dehydrogenase x Kan Kanamycin KDPG 2-keto-3-deoxy-6-phosphogluconate KGD ø-ketoglutarate dehydrogenase LB Luria-Bertani MDH Malate dehydrogenase mRNA Messenger RNA MCP Methyl-acceptingchemotaxisprotein NAD.Ì Nicotinamideadeninedinucleotide NADPr Nicotinamide adenine dinucleotide phosphate Nm Neomycin NMR Nuclear magnetic resonance ODcoo Optical density at 600 nm ORF Open reading frame PCK Phosphoenolpyruvate carboxykinase PCR Polymerase chain reaction PFK Phosphofructokinase PGDH 6-phosphogluconatedehydrogenase PHB Polyhydroxybutyrate POD Pyruvateorthophosphatedikinase PP Pentose phosphate PYC Pyruvate carboxylase X Rf Rifampicin RNA Ribonucleic acid SDH Succinatedehydrogenase Sm Streptomycin Tc l.etracycline TCA Tricarboxylic acid TME Triphosphopyridine-nucleotidedependentmalic enzyme TPI Triose phosphate isomerase IRNA Transfer RNA TY Tryptoneyeast VMM Vincent's minimal medium XI Chapter I Introduction Sinorhizobium melilorl is a Gram-negative bacterium which is studied as a model sys- tenr for plant-microbe symbiosis. S. meliloti lives a saproph¡ic lifestyle and has the ability to form a symbiotic
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