Probing the Sinorhizobium meliloti-alfalfa symbiosis using temperature-sensitive and impaired-function citrate...

To study the role of the decarboxylating leg of the bacterial TCA cycle in symbiotic nitrogen fixation, we used DNA shuffling and localized random polymerase chain reaction mutagenesis to construct a series of temperature-sensitive and impaired-function mutants in the Sinorhizobium meliloti Rm104A14 citrate synthase (gltA) gene. Reducing citrate synthase (CS) activity by mutation led to a corresponding decrease in the free-living growth rate; however, alfalfa plants formed fully effective nodules when infected with mutants having CS activities as low as 7% of the wild-type strain. Mutants with approximately 3% of normal CS activity formed nodules with lower nitrogenase activity and a mutant with less than 0.5% of normal CS activity formed Fix- nodules. Two temperature-sensitive (ts) mutants grew at a permissive temperature (25 degrees C) with 3% of wild-type CS activities but were unable to grow on minimal medium at 30 degrees C. Alfalfa plants that were inoculated with the ts mutants and grown with a root temperature of 20 degrees C formed functional nodules with nitrogenase activities approximately 20% of the wild type. When the roots of plants infected with the ts mutants were transferred to 30 degrees C, the nodules lost the ability to fix nitrogen over several days. Microscopic examination of these nodules revealed the loss of bacteroids and senescence, indicating that CS activity was essential for nodule maintenance.