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Aspartate transport by the Dct system in Rhizobium leguminosarum negatively affects nitrogen-regulated operons

Colm J. Reid^1,

School of Animal and Microbial Sciences, University of Reading, Whiteknights, PO Box 228, Reading RG6 2AJ, UK

²Author for correspondence: Philip S. Poole. Tel: +44 1734 318895. Fax: +44 1734 316671. e-mail: p.s.poole@reading.ac.uk

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 glnll was repressed by aspartate. The negative regulatory effect on both the Aap and glnll 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 K_m for succinate of 5 µM and an apparent K_i 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.

Present address: Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, UK.

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