title: Aspartate transport by the Dct system in Rhizobium leguminosarum negatively affects nitrogen-regulated operons.
abstract: Amino acid uptake by the general amino acid permease (Aap) of Rhizobium leguminosarum strain 3841 was severely reduced by the presence of aspartate in the growth medium when glucose was the carbon source. The reduction in transport by the Aap appeared to be caused by inhibition of uptake and not by transcriptional repression. However, as measured with lacZ fusions, the Ntr-regulated gene glnII was repressed by aspartate. The negative regulatory effect on both the Aap and glnII was prevented by mutation of any component of the dicarboxylate transport (Dct) system or by the inclusion of a C4- dicarboxylate in the growth medium, including the non-metabolizable analogue 2-methylsuccinate. As measured by total uptake and with a dctA-lacZ fusion, aspartate was an efficient inducer of the Dct system, but slightly less so than succinate alone or succinate and aspartate together. Thus, aspartate does not cause overexpression of DctA leading to improper regulation of other operons. Transport measurements revealed that the Dct system has an apparent Km for succinate of 5 microM and an apparent Ki for aspartate inhibition of succinate uptake of 5 mM. These data imply that the Dct-mediated accumulation of aspartate causes an unregulated build-up of aspartate or a metabolic product of it in the cell. This accumulation of aspartate is prevented either by mutation of the dct system or by the presence of a higher affinity substrate that will reduce access of aspartate to the carrier protein. Elevation or disruption of the intracellular aspartate pool is predicted to disrupt N-regulated operons and nitrogen fixation.
authors.affiliation: School of Animal and Microbial Sciences, University of Reading, Whiteknights, UK.
authors.forename: C J
authors.initials: CJ
authors.lastname: Reid
authors.forename: D L
authors.initials: DL
authors.lastname: Walshaw
authors.forename: P S
authors.initials: PS
authors.lastname: Poole
mesh_terms: D026905:Amino Acid Transport Systems; D001224:Aspartic Acid / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001426:Bacterial Proteins; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003470:Culture Media / Q000494:pharmacology; D029382:Dicarboxylic Acid Transporters; D003998:Dicarboxylic Acids / Q000494:pharmacology; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005947:Glucose / Q000378:metabolism; D005974:Glutamate-Ammonia Ligase / Q000235:genetics; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000235:genetics; D016254:Mutagenesis, Insertional; D009586:Nitrogen Fixation / Q000235:genetics; D011401:Promoter Regions, Genetic; D016961:Rhizobium leguminosarum / Q000235:genetics* / Q000378:metabolism*; D017384:Sequence Deletion; D013386:Succinates / Q000494:pharmacology; D019802:Succinic Acid; D014158:Transcription, Genetic
chemical_list: D026905:Amino Acid Transport Systems; D001426:Bacterial Proteins; D002352:Carrier Proteins; D003470:Culture Media; D029382:Dicarboxylic Acid Transporters; D003998:Dicarboxylic Acids; D026901:Membrane Transport Proteins; D013386:Succinates; C073786:dctA protein, Sinorhizobium meliloti; D001224:Aspartic Acid; D019802:Succinic Acid; C083818:glutamine synthetase 2; D005974:Glutamate-Ammonia Ligase; D005947:Glucose; C041105:monomethyl succinate