Biochemical and Genetical Analysis of Rhizobium meliloti Mutants Defective in C4-dicarboxylate Transport

SUMMARY: Inhibition studies with free-living cells of Rhizobium meliloti2011 showed that succinate, fumarate and malate are transported via a common transport system. It is an active process that is inducible by succinate. The apparent K m for succinate uptake in free-living cells is 5·3 μm. Seven Tn5-induced mutants that did not grow on succinate, fumarate or malate lacked the C4-dicarboxylate transport system in the free-living state (Detfl-). Five of these mutants (RMS11, RMS16, RMS17, RMS24, RMS118) induced nodules on alfalfa with about half the nitrogen-fixation activity of the wild-type (Fixred). The remaining two mutants (RMS420 and RMS938) gave rise to small, white and ineffective nodules on alfalfa (Fix-). These results correlated with the dicarboxylate transport rates of bacteroids isolated from appropriate nodules: bacteroids isolated from nodules induced by the Fixred mutants transported succinate or malate at about 30-50% of the wild-type rate (Dctsred), whereas bacteroids isolated from nodules induced by Fix-mutants showed no uptake activity (Dcts-). From an R. meliloti 2011 bank three cosmids were identified which complemented different Dctfl- mutants. Complemented Dctfl- mutants could grow again on agar containing succinate or malate. In the case of Fix- mutants complementation resulted in effective nodules. The cosmids pRmSC101 and pRmSC102 complemented the Fix- mutant RMS420. A third cosmid, pRmSC121, complemented the second Fix- mutant RMS938 and nearly all of the Fixred mutants. For the Fix- mutant RMS420 the Tn5 insertion site was found to be located on a 15·3 EcoRI fragment which is also part of the complementing cosmid pRmSC102.