Molecular and expression analysis of the Rhizobium meliloti phosphoenolpyruvate carboxykinase (pckA) gene.
The pckA gene of Rhizobium meliloti, encoding phosphoenolpyruvate carboxykinase, was isolated from a genomic cosmid library by complementation of the succinate growth phenotype of a Pck- mutant. The gene region was mapped by subcloning and Tn5 insertion mutagenesis. The DNA sequence for a 2-kb region containing the structural gene and its promoter was determined. The pckA gene encodes as 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 with a pckA::lacZ gene fusion indicated that the pckA gene was strongly induced at the onset of stationary phase in complex medium. When defined carbon sources were tested, the expression level of the pckA gene was found to be high when cells were grown in minimal media with succinate or arabinose as the sole carbon source but almost absent when glucose, sucrose, or glycerol was the sole carbon source. Glucose and sucrose were not found to strongly repress pckA induction by succinate.
Osterås
M
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Department of Biology, McMaster University, Hamilton, Ontario, Canada.
Driscoll
B T
BT
Finan
T M
TM
D000595:Amino Acid Sequence; D001483:Base Sequence; D001692:Biological Transport; D003001:Cloning, Molecular; D003998:Dicarboxylic Acids / Q000378:metabolism; D004790:Enzyme Induction; D004794:Enzyme Repression; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial / Q000235:genetics*; D005947:Glucose / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D016962:Sinorhizobium meliloti / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D013386:Succinates / Q000378:metabolism; D019802:Succinic Acid; D013395:Sucrose / Q000378:metabolism; D014158:Transcription, Genetic
D003998:Dicarboxylic Acids; D013386:Succinates; D013395:Sucrose; D019802:Succinic Acid; D010729:Phosphoenolpyruvate Carboxykinase (GTP); D005947:Glucose