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Genetics, Vol 147, 1521-1531, Copyright © 1997
INVESTIGATIONS |
Genetic Analysis of Mutations Affecting pckA Regulation in Rhizobium (Sinorhizobium) meliloti
M. {Oslash}steras, SAP. O'Brien and T. M. Finan
Present address: Laboratoire de Biologie Vegetale et Microbiologie, URA CNRS 1114, Universite de Nice Sophia Antipolis, 06108 Nice, France.
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 N(2)-fixing root nodules on alfalfa, and the major energy sources supplied to the bacteria within these nodules are C(4)-dicarboxylic acids such as malate and succinate. R. meliloti cells growing in glucose minimal medium show very low 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 that pckA is expressed in media containing the non-inducing carbon sources lactose and glucose. Genetic and phenotypic analysis allowed us to differentiate at least four rpk mutant classes that map to different locations on the R. meliloti chromosome. The wild-type locus corresponding to one of these rpk loci was cloned by complementation, and two Tn5 insertions within the insert DNA that no longer complemented the rpk mutation were identified. The nucleotide sequence of this region revealed that both Tn5 insertions lay within a gene encoding a protein homologous to the GalR/LacI family of transcriptional regulators that are involved in metabolism.
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