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The General Amino Acid Permease of Rhizobium Leguminosarum Biovar Viciae

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The General Amino Acid Permease of Rhizobium Leguminosarum Biovar Viciae The reasonable man attempts to adapt himself to suit the world: the unreasonable one persists in trying to adapt the world to suit himself. Therefore all progress depends upon the unreasonable man. George Bernard Shaw UNIVERSITY OF READING School of Animal and Microbial Sciences The General Amino Acid Permease of Rhizobium leguminosarum biovar viciae by David L. Walshaw Submitted in partial fulfilment of the requirement for the degree of Docter of Philosophy 1995 I declare that this thesis is my own account of my research and that this work has not previously been submitted for a degree at any University. However, I would like to acknowledge the help I received from the undergraduate project students Adam Wilkinson and Mathias Mondy in restriction mapping the cosmid pRU3004, under my joint supervision with Dr P.S.Poole. David Walshaw TABLE OF CONTENTS Page CHAPTER 1 LITERATURE REVIEW 1 1.1 INTRODUCTION 2 1.1.1 Rhizobia 2 1.1.2 Nodule formation and structure 2 1.2 BACTEROID METABOLISM 5 1.2.1 Carbon sources supplied to the bacteroid 5 1.2.2 The TCA cycle in the bacteroid 8 1.2.3 The role of poly-β-hydroxybutyrate biosynthesis 13 1.2.4 The malate-aspartate shuttle 14 1.2.5 Other roles of amino acids in bacteroid metabolism 19 1.3 AMINO ACID TRANSPORT IN BACTERIA 27 1.4 ABC TRANSPORTERS 29 1.4.1 Overall structure of ABC transporters 29 1.4.2 The transmembrane domains 32 1.4.3 The ATP-binding domains 35 1.4.4 Periplasmic binding proteins 40 1.4.5 Mechanism of solute translocation 44 1.4.6 The role of binding protein-dependent transporters 49 1.4.7 Regulation of ABC transporters 49 1.5 AMINO ACID TRANSPORT IN RHIZOBIUM 51 1.6 REGULATION INVOLVING NTRC 53 CHAPTER 2 MATERIALS AND METHODS 58 2.1.1 Bacterial strains 59 2.1.2 Culture conditions 68 2.1.3 DNA and genetic manipulations 68 2.1.4 Mutagenesis 69 2.1.5 Transport Assays 70 2.1.6 Isolation of periplasmic fractions and protein gel electrophoresis 71 2.1.7 Protein binding assays 71 2.1.8 Enzyme assays 72 2.1.9 Metabolite excretion assays 73 2.1.10 Intracellular concentrations 74 Page 2.1.11 Protein determination 75 2.1.12 Plant Assays 75 CHAPTER 3 THE CLONING AND CHARACTERIZATION OF THE GENERAL AMINO ACID PERMEASE OF RHIZOBIUM LEGUMINOSARUM STRAIN 3841 76 3.1 INTRODUCTION 77 3.2 RESULTS 78 3.2.1 Isolation of cosmid pRU3024 carrying the general amino acid permease genes of Rhizobium leguminosarum strain 3841 78 3.2.2 Restriction mapping, sub-cloning and mutational analysis of pRU3024 80 3.2.3 Nucleotide sequence of the 5.4kb MluI-ClaI fragment of pRU135 85 3.2.4 Coding regions of the nucleotide sequence from pRU189 97 3.2.5 Other features of the nucleotide sequence from pRU189 106 3.2.6 Mutation of the general amino acid permease 107 3.2.7 Mapping of promoter sites in the aap operon by complementation analysis 111 3.2.8 Transcription levels of aap genes 113 3.2.9 Amino acid uptake in strains RU542, RU543, RU634 and RU636 114 3.2.10 Growth of strain RU543 on amino acids as sole source of carbon and nitrogen 116 3.2.11 Amino acid uptake in strain RU640 118 3.2.12 Expression of the R. leguminosarum general amino acid permease in E. coli 118 3.2.13 Physical properties of the aapJ gene product 120 3.2.14 Effect of aapJ on amino acid uptake in 3841 122 3.2.15 Effect of aapQMP on amino acid uptake in strains 3841 123 3.2.16 Specificity of the aapJ gene product 124 3.2.17 Substrate-binding activity of AapJ 125 Page 3.2.18 Amino acid exchange 130 3.2.19 Plant properties of strains RU542, RU543, RU634, and RU636 143 3.2.20 Nucleotide sequence of the 0.8kb BamHI fragment of pRU3024 144 3.2.21 Amino acid transport in strain RU632 147 3.2.22 Plant properties of RU632 149 3.2.23 Nucleotide sequence adjacent to the transposon in cosmids pRU3053, pRU3082, pRU3083, pRU3084, pRU3085 and pRU3086 150 3.2.24 Amino acid transport in metC mutants of strain 3841 154 3.3 DISCUSSION 155 CHAPTER 4 NITROGEN REGULATION OF THE GENERAL AMINO ACID PERMEASE OF RHIZOBIUM LEGUMINOSARUM STRAIN 3841 161 4.1 INTRODUCTION 162 4.2 RESULTS 163 4.2.1 Effect of the metC-aapJ intergenic region on growth of strain 3841 163 4.2.2 Effect of nitrogen supply on the transcription of aapJQM 164 4.2.3 Amino acid uptake in strain RU929 169 4.2.4 Effect of nitrogen supply on the transcription metC and cysE 170 4.2.5 Sequence analysis of the metC-aapJ intergenic region 173 4.3 DISCUSSION 175 CHAPTER 5 INTER-REGULATION OF THE TCA CYCLE AND THE GENERAL AMINO ACID PERMEASE OF R. LEGUMINOSARUM STRAIN 3841. 177 5.1 INTRODUCTION 178 5.2 RESULTS 179 Page 5.2.1 Aspartate resistant mutants of R. leguminosarum strain 3841 179 5.2.2 Growth of strains RU116, RU118, RU137 and RU156 on succinate and glucose 183 5.2.3 Amino acid transport in strains RU116 and RU156 184 5.2.4 Transductional analysis of strains RU116, RU118, RU137 and RU156 186 5.2.5 Nucleotide sequence adjacent to the transposon in strains RU116, RU137 and RU156 187 5.2.6 Activity of TCA cycle enzymes in strains RU116, RU118, RU137 and RU156 194 5.2.7 Growth of strains RU116, RU137 and RU156 on arabinose 195 5.2.8 Complementation of strain RU156 196 5.2.9 Effect of pRU3004 on aspartate transport in strains RU116, RU137, and RU156 197 5.2.10 Southern blot of pRU3004 against RU116, RU137, RU156 chromosomal DNA 198 5.2.11 Restriction mapping, sub-cloning and mutation of pRU3004 199 5.2.12 Genes carried by pRU3004 201 5.2.13 β-galactosidase activities from pRU3004 mutants 210 5.2.14 TCA cycle enzyme activities in sucCDAB mutants of strain 3841 210 5.2.15 Mapping of promoter sites in pRU3004 211 5.2.16 Amino acid excretion by strains RU116, RU156 and RU543 215 5.2.17 Intracellular concentrations of α-ketoglutarate and glutamate in strains RU116 and RU156 220 5.2.18 Transcription of the aap operon in sucDA mutants of strain 3841 222 5.2.19 Plant properties of strains RU116, RU137 and RU156 224 5.3 DISCUSSION 226 Page CHAPTER 6 FINAL DISCUSSION 231 6.1.1 The general amino acid permease of Rhizobium leguminosarum 232 6.1.2 TCA cycle enzymes in Rhizobium leguminosarum 237 6.1.3 Methionine biosynthetic enzymes in Rhizobium leguminosarum 238 6.1.4 Future work 239 REFERENCE 241 S ABSTRACT The four genes, aapJQMP, encoding the general amino acid permease of R. leguminosarum strain 3841 have been cloned, sequenced, and shown to be transcribed as a single operon. Sequence homology data indicate that these genes encode the components of an ABC transporter. However, the periplasmic binding protein, AapJ, and the two integral membrane components, AapQ and AapM, are significantly larger than the equivalent components of previously described ABC transporters of amino acids. The strong homology of these proteins to sequence from the Escherichia coli genome sequencing project suggests that E. coli may possess a previously unreported general amino acid permease. Transcription of the aap operon has been shown to be negatively regulated by NtrC in response to nitrogen supply. Mutation of any one of the aap genes resulted in a reduction in the uptake by strain 3841 of a range of amino acids, including aliphatic amino acids such as leucine and alanine, and polar amino acids such as glutamate, aspartate and histidine. Over expression of the aap operon resulted in a marked increase in the uptake of all the amino acids tested. The results of experiments to investigate the effect of mutation and over expression of aap genes on amino acid exchange by strain 3841, appear to indicate that the general amino acid permease facilitates both uptake and efflux of amino acids. The involvement of the general permease in amino acid efflux is also indicated by the reduced glutamate excretion during growth on glucose/NH4Cl/aspartate, exhibited by an aapJ mutant of strain 3841. An attempt to isolate general amino acid permease mutants on the basis of resistance to a toxic concentration of aspartate, led to the discovery that mutation of genes encoding the TCA cycle enzymes α-ketoglutarate dehydrogenase and succinyl-CoA synthetase causes almost total abolition of uptake by the general amino acid permease of strain 3841. This effect has been shown not to be due to regulation of aap gene expression at the transcriptional level. Nor does it appear to be the result of substrate-inhibition of the transporter, despite the fact that α-ketoglutarate dehydrogenase and succinyl- CoA synthetase mutants are found to accumulate and excrete glutamate. It is therefore proposed that the general amino acid permease may be subject to post-translational modification. The chromosomal region containing contiguous genes that code for malate dehydrogenase, succinyl-CoA synthetase and α-ketoglutarate dehydrogenase, has been cloned and partially sequenced. Mutants in any one of the components of the general amino acid permease were found to induce pea root nodules that reduce acetylene as effectively as those of the wild-type strain. α-Ketoglutarate dehydrogenase and succinyl- CoA synthetase mutants of strain 3841 formed ineffective nodules. CHAPTER 1 LITERATURE REVIEW 1 1.1 INTRODUCTION 1.1.1 Rhizobia Bacteria of the genus Rhizobium stimulate leguminous plants to develop root nodules which the bacteria infect and inhabit.
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