MOLECULAR AND EXPRESSION ANALYSIS OF THE RHIZOBIUM-MELILOTI PHOSPHOENOLPYRUVATE CARBOXYKINASE (PCKA) GENE

The pckA gene of Rhizobium meliloti, encoding phosphoenolpyruvate carb oxykinase, was isolated from a genomic cosmid library by complementati on of the succinate growth phenotype of a Pck(-) mutant, The gene regi on was mapped by subcloning and Tn5 insertion mutagenesis. The DNA seq uence for a 2-kb region containing the structural gene and its promote r was determined. The pckA gene encodes a 536-amino-acid protein that shows homology with other ATP-dependent Pck enzymes, The promoter was identified following primer extension analysis and is similar to sigma (70)-like promoters. Expression analysis,vith a pckA::lacZ gene fusion indicated that the pckA gene was strongly induced at the onset of sta tionary phase in complex medium, When defined carbon sources were test ed, the expression level of the pckA gene was found to be high when ce lls were grown in minimal media with succinate or arabinose as the sol e carbon source but almost absent when glucose, sucrose, or glycerol w as the sole carbon source. Glucose and sucrose were not found to stron gly repress pckA induction by succinate.