ASPARTATE TRANSPORT BY THE DCT SYSTEM IN RHIZOBIUM-LEGUMINOSARUM NEGATIVELY AFFECTS NITROGEN-REGULATED OPERONS

Amino acid uptake by the general amino acid permease (Aap) of Rhizobiu m leguminosarum strain 3841 was severely reduced by the presence of as partate in the growth medium when glucose was the carbon source. The r eduction in transport by the Aap appeared to be caused by inhibition o f uptake and not by transcriptional repression. However, as measured w ith lacZ fusions, the Ntr-regulated gene glnII was repressed by aspart ate, The negative regulatory effect on both the Aap and glnII was prev ented by mutation of any component of the dicarboxylate transport (Dct ) system or by the inclusion of a C-4-dicarboxylate in the growth medi um, including the non-metabolizable analogue 2-methylsuccinate. As mea sured by total uptake and with a dctA-lacZ fusion, aspartate was an ef ficient inducer of the Dct system, but slightly less so than succinate alone or succinate and aspartate together. Thus, aspartate does not c ause overexpression of DctA leading to improper regulation of other op erons. Transport measurements revealed that the Dct system has an appa rent K-m for succinate of 5 mu M and an apparent K-i for aspartate inh ibition of succinate uptake of 5 mM. These data imply that the Dct-med iated accumulation of aspartate causes an unregulated build-up of aspa rtate or a metabolic product of it in the cell. This accumulation of a spartate is prevented either by mutation of the dct system or by the p resence of a higher affinity substrate that will reduce access of aspa rtate to the carrier protein. Elevation or disruption of the intracell ular aspartate pool is predicted to disrupt N-regulated operons and ni trogen fixation.