GENETIC-ANALYSIS OF MUTATIONS AFFECTING PCKA REGULATION IN RHIZOBIUM (SINORHIZOBIUM) MELILOTI

The enzyme phosphoenolpyruvate carboxykinase (Pck) catalyzes the first step in the gluconeogenic pathway in most organisms. We are examining the genetic regulation of the gene encoding Pck, pckA, in Rhizobium ( now Sinorhizobium) meliloti. This bacterium forms Ne-fixing root nodul es on alfalfa, and the major energy sources supplied to the bacteria w ithin these nodules are C-4-dicarboxylic acids such as malate and succ inate. R meliloti cells growing in glucose minimal medium show very lo w pckA expression whereas addition of succinate to this medium results in a rapid induction of pckA transcription. We identified spontaneous mutations (rpk) that alter the regulation of pckA expression such tha t pckA is expressed in media containing the non-inducing carbon source s lactose and glucose. Genetic and phenotypic analysis allowed us to d ifferentiate at least four rpk mutant classes that map to different lo cations on the R meliloti chromosome. The wild-type locus correspondin g to one of these rpk loci was cloned by complementation, and two Tn5 insertions within the insert DNA that no longer complemented the rpk m utation were identified. The nucleotide sequence of this region reveal ed that both Tn5 insertions lay within a gene encoding a protein homol ogous to the GalR/LacI family of transcriptional regulators that are i nvolved in metabolism.