3t' (O' COMPARATIVE STUDIES OF OXALYL.COA DECARBOXYLASE PRODUCED BY SOIL AND RUMINAL BACTERIA Thesis Submitted for the degree of Master of Agricultural Science in The University of Adelaide Faculty of Agricultural and Natural Resource Sciences by STEPHEN BOTTRILL November 1999 I I Table of Contents List of Figures VI List of Tables VM Abstract IX Acknowledgements XII Ståtement XIII List of Abbreviations XTV Chapær 1. Liærature Review 1 1.1 Introduction. 1 1.2 Exogenous Sources of Oxalates. 1 1.3 Endogenous Sources of Oxalate. 5 1.4 Poisoning. 9 1.4.1 Acute Poisoning" 10 1.4.2 Subacute Poisoning. 11 1.4.3 Chronic Poisoning. t2 1.4.4 SymPtoms in Humans. 14 1.4.5 Treatment of Poisoning. I4 1.4.6 Management to Prevent Poisoning. 15 1.5 Oxalate-Degrading Microorganisms. 18 1.6 Bacterial Classification 22 1.7 Pathways of Oxalate Degradation. 24 1.8 Formate in the Rumen. 27 1.9 Aims and Objectives. 29 Chapær 2. Materials and Methods 31 2.1 Materials 31 2.1.1 Chemicals 3r 2.1.2 EquiPment 31 2.I.3 Bacterial Strains and Plasmids 32 TI 2.1.4 Composition of Media 34 2.1.4.I Oxalate-Containing Media 34 2.1.4.I.1Liquid 34 2.1.4.1.2 Solid 34 2.1.4.2 O mlob act er formi g enes Media 35 2.I.4.2.I Trace Metals Solution 35 2.I.4.2.2 Medium A 35 2.1.4.2.3 Medium B 36 2.I.4.3 Luria-Bertani (LB) Broth 36 2.I.4.4 SOC Medium 37 2.2 Methods 37 2.2 -I Growth conditions 37 2.2.2 Isolation of oxal ate- de gradin g s oil bacteria 37 2.2.3 Characterisation of soil isolaæs 38 2.2.3.1 MicroscoPY 38 2.2.3.2 Gram stain 38 2.2.3.3 Carbon source utilisation 40 2.2.4.4 Volatile fatty acid anaþsis 40 2.2.4 PhenoVchloroform extractions 4l 2.2.5 Ethanol preciPitations 4l 2.2.6 Chromosomal DNA preparation 42 2.2.7 Plasmid DNA minipreparations 42 2.2.8 Restriction digests 43 2.2.9 Ligations 43 2.2.10 ComPetent cells 44 2.2.I I ElectroPoration 44 2.2.12 Selection for transformants 44 2.2.13 Agarose gel electrophoresis 45 2.2.I4 Conjugation 46 m 2.2.1 5 Southern transfer 46 2.2.16 Prehybridisation 47 2.2.17 Oligolabelling 48 2.2.18 Hybridisation 48 2.2.L9 Washing 48 2.2.19 Stripping the membrane 49 2.2.20 Nested deletions 49 2.2.21DNA sequencing 50 2.2.22 Dot blots 5l 2.2-23 Polymerase chain reaction 51 2.2.24 Restriction Fragment Length Polymorphisms 52 2.2.25 Polyclonal antibody production 53 2.2.26 Enzyme-linked immunosorbent assay 54 2.2.27 Western transfer 57 2.2.28 RNA preparation 58 2.2.29 Northern transfer 60 2.2.30 Polyacrylamide gel electrophoresis 62 2.2.30.I Denaturing (Protein) 62 2.2.30.2 Non-denaturing (protein) 64 2.2.30.3 Denaturing (DNA) 64 2.2.31 Synthesis of OxalYl-CoA 66 2.2.32 Free thiol test 66 2.2.33 Enzyme assays 67 2.2.33.t Oxalyl-CoA decarboxylase 67 2.2.33.2 Formate dehYdrogenase 68/ 2.2.33 .3 Glyoxylate dehydro genase 68 2.2.34 Colony lifts 69 2.2.35 Cell-free extracts 69 2.2.36 Protein estimation 70 IV 2.2 -37 PaPer chromatograPhY 7T 2.2.38 Cosmid Packaging 7T Chapter 3. Isolation and characterisation of Oxalaæ-Degrading Bacteria 73 3.1 Introduction 73 3.2 Results 74 3.2.1 Isolation of oxalate-Degrading Bacteria from the Soil 74 3 .2.2 Chat acteris atio n 76 3.2.2.I B iochemical AnalYsis 76 3.2.2.2 Genetic Analysis 76 3.2.2.2.I Chromosomal DNA Homology 76 3.2.2.2.2 Chrom o som al DNA Restriction Profiles 77 3.2.2.2.3 Restriction Fragment Length PolYmorPhisms 77 3.2.2.2.4 Sequence Analysis of the 165 Subunit of rRNA 81 3.2.3 Growth Curves 81 83 3 .2.4 Oxalate Tolerance 3.2.5 Substrate Utilisation 84 3.2.6 Volatile Fatty Acid Analysis 84 3.3 Discussion 86 Chapter 4.Enzyme Systems in the Metabolism of Oxalate 9l 4.1 Introduction 91 4.2 Results 92 4.2.lDetermination of Assay conditions for Paracocc¿rs sp. OxaIyl-CoA Decarboxylase 92 4.2.I.1 Assay Requirements 93 4.2.1.2 pH Optimum 93 4.2.2 Comparison of Enzyme Systems in Paracocc¿'s sp' and P s eu do nnna s o xalati c us 94 V 4.2.2.L Oxalyl-CoA Decarboxylase 94 4.2.2.2 Formate Dehydro genase 95 4.2.3 Ind:uction of P. oxalati cus Oxalyl-CoA Decarboxylase 95 4.2.4 Determination of K¡n and Y yn6ç 96 4.3 Discussion 97 Chapter 5. Cloning of the Genes for Oxalaæ Decarboxylation 101 5.l lntroduction 101 5.2 Results 103 5.2.I Plate AssaYs 103 5.2.2 Antlbody Probes 106 5.2.3 Generation of Ox- Mutants 106 5.2.4 Generation of DNA Probes from P. oxalaticus 108 5.2.4.1 Transposon Mutagenesis 108 5-2-4.2 Polymerase Chain Reaction 109 5.2.5 Sequencing rr4 5.2.5.L Mapping LT4 5.2.5.2 Nested Deletions tl4 5.2.5.3 Sequence Data 115 5.2.6 Generation of DNA probes from O. formigenes rt7 5.3 Discussion rt7 Chapter 6. General Discussion and Future Studies t26 Appendix 1 131 Appendix 2 135 Appendix 3 138 References 139 VI List of Figures Figure 3.1 Halo formation by OxD on solid Bhat and Barker medium. 75 Figure 3.2Dotblot of oxalate-degrading soil isolates. 78 Figure 3.3 Chromosomal DNA Restriction Prohles' 79 Figure 3.4 Restriction fragment length polymorphisms. 80 Figure 3.5 Growth of soil isolates in Bhat and Barker oxalate medium. 82 Figure 3.6 Growth of OxD in Bhat and Barker oxalate medium. 82 Figure 3.7 Oxalate tolerance. 83 Figure 4.1. Opúmum pH for Paracocc¿rs sp. oxalyl-CoA decarboxylase activity 94 Figure 4.2. OxaIyl-CoA decarboxylase activity in crude cell-free extracts of Pseudomonas oxnlaticas and Paracoccus sp- 95 Figure 4.3. Formate dehydrogenase activity in crude cell-free extracts of Pseudamonas oxnlaticus and Paracoccus sp- 95 'tn Figure 4.4. Induction of oxalyl-CoA decarboxylase activity Pseudomonas oxalaticus and Paracoccus sP. 96 Figure 4.5. Lineweaver-Burk plot for the oxalyl-CoA decarboxylase of Paracoccus sP. 97 Figure 5.1 Pseudomanas oxalatícus DNA partially digested with Bam HI. 104 Figure 5.2 Randomly picked cosmids from a library of Pseudomonas oxalaticus DNA cloned in PHC79. 105 Figure 5.3 Western immunoblot of bacterial cell-free extracts- t07 Figure 5.4 Survival of Pseudomonas oxnlaticus aftÊr exposure to ultra violet radiation. 108 VII Figure 5.5 Southern blot of the integration of transposonTnl732 into the chromosome of P seudomona.s oxalaticus. 110 Figure 5.6 PCR of Pseudomnnas oxalatícus DNA. 111 Figure 5.7 Southern transfer of Pseudomonas oxalatic¿¿s DNA probed with a Ikb Pseudomonas oxalaticus PCR product. rt2 Figure 5.8 Colony ltft of. Escherichia coli colonies- 113 Figure 5.9 Restriction map of the Hind Itr DNA fragment of Pseudomonas oxnlaticus- rt4 Figure 5.10 Nested deletions. 1r6 Figure 5.11 Testing of PCR probes from Pseudomanas oxalaticus. 118 Figure 5.12 Testing of PCR probes ftom Oxalobacter formígenes. 119 Figure 5.13 Southern transfer of Pseudomanas oxalaticus DNA probed with a 1 kb Oxalobacter formigenes PCR product. t20 VItr List of Tables 33 Table 2.L. B acteial Strains 33 Table 2.2. Plasmids 33 Table 2.3. Antibiotics 4t Table 2.4. VFA Analysis Conditions. Table 3.1 Characærisation of OxD by the Instituæ of Veterinary and Medical 76 Science. Table 3.2 OxD substrate utilisation and comparison with Pseudnmonas 85 diminuta and P s eudomonas oxal'aticus' Table 4.1. Cofactors for the decarboxylation of oxalate by oxalyl-CoA decarboxylase derived from soil isolaæ Paracoccus sp' 93 IX Abstract Oxalic acid is an organic dicarboxylic acid which has a marked affinity for calcium salts are sodium, and magnesium, the salts of which are insoluble in water. Its principle plants, some of potassium, and ammonium. The salts of oxalic acid are found in a variety of pastures which form the dominant pasture species. Stock losses as a result of grazing such have been responsible for are common. Oxalate-producing fungi have also been reported to stock losses. usually Oxalate poisoning of stock can take either of two forms. Acute poisoning plants' occufs when hungry stock are placed on pasture dominated by oxalate-containing deficient in Reduction of rumen microbial activity, usually due to fasting, as well as diets dose as a result calcium, predispose to toxicity. Death usually occurs 4- 12 hours after a lethal 2 or 3 days. of severe metabolic disturbance, although some sheep may linger on for gtazing Oxalis pes- Chronic oxalate toxicity is common in sheep which have been adapæd to of the acid caprae (soursob). They develop a high degree of tolerance to high daily intakes of the oxalate oxalate contained by the plants. However, there is ofæn low-level absorption with progressive damage to the kidneys as a result of deposition of masses of calcium develop oxalate crystals.