Molecular mechanism of the regulation of Bacillus subtilis gltAB expression by GltC.
In Bacillus subilis, glutamate synthase, a major enzyme of nitrogen metabolism, is encoded by the gltAB operon. Significant expression of this operon requires the activity of GltC, a LysR-family protein, encoded by the divergently transcribed gene. We purified a soluble, active form of GltC and found that it requires alpha-ketoglutarate, a substrate of glutamate synthase, to fully activate gltA transcription in vitro, and that its activity is inhibited by glutamate, the product of glutamate synthase. GltC regulated gltAB transcription through binding to three dyad-symmetry elements, Box I, Box II and Box III, located in the intergenic region of gltC and gltA. Free GltC bound almost exclusively to Box I and activated gltAB transcription only marginally. Glutamate-bound GltC bound to Box I and Box III, and repressed gltAB transcription. In the presence of alpha-ketoglutarate, GltC bound to Box I and Box II, stabilized binding of RNA polymerase to the gltA promoter, and activated gltAB transcription. The binding of GltC to Box II, which partially overlaps the -35 region of the gltA promoter, seems to be essential for activation of the gltAB operon. Due to the high concentration of glutamate in B. subtilis cells grown under most conditions, alterations of the concentration of alpha-ketoglutarate seem to be crucial for modulation of GltC activity and gltAB expression.
Picossi
Silvia
S
Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA 02111, USA.
Belitsky
Boris R
BR
Sonenshein
Abraham L
AL
D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D018983:DNA Footprinting; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D005970:Glutamate Synthase / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000096:biosynthesis; D006639:Histidine / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000494:pharmacology; D008957:Models, Genetic; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D012097:Repressor Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects
D001426:Bacterial Proteins; C095882:GltC protein, Bacillus subtilis; C471213:His-His-His-His-His-His; D007656:Ketoglutaric Acids; D050505:Mutant Proteins; D009842:Oligopeptides; D012097:Repressor Proteins; D015534:Trans-Activators; D018698:Glutamic Acid; D006639:Histidine; D005970:Glutamate Synthase; D012321:DNA-Directed RNA Polymerases; D003850:Deoxyribonuclease I