The formation of phosphoenolpyruvate (PEP) is a major step in the gluconeogenic pathway in which tricarboxylic acid (TCA) cycle intermediates are converted to hexose sugars. In
Rhizobium(now Sinorhizobium) melilotithis step is catalysed by the enzyme PEP carboxykinase (PCK) which converts oxaloacetate to PEP. R. melilotiPck-mutants grow very poorly with TCA cycle intermediates as the sole source of carbon. Here, the isolation and mapping of suppressor mutations which allow Pck-mutants to grow on succinate and other TCA cycle intermediates is reported. Tn 5insertions which abolished the suppressor phenotype and mapped to the suppressor locus were located within the pod gene encoding pyruvate orthophosphate dikinase (PPDK). Strains carrying suppressor mutations had increased PPDK activity compared to the wild-type. The suppressor phenotype was dependent on the combined activities of malic enzyme and PPDK, which thus represent an alternative route for the formation of PEP in R. meliloti.PPDK activity was not required for symbiotic N2 fixation.