ArgR-Independent Induction and ArgR-Dependent Superinduction of theastCADBEOperon inEscherichia coli

Abstract

For Escherichia coli, growth in the absence of ammonia is termed nitrogen limited and results in the induction of genes that assimilate other nitrogen sources, a response mediated by 54 and nitrogen regulator I (NR I , also called NtrC). The astCADBE operon, which is required for growth with arginine as the sole nitrogen source, is moderately expressed during general nitrogen limitation and maximally expressed in the presence of arginine. The operon is also induced in stationary phase. Primer extension analysis of E. coli revealed the presence of a 54 -dependent promoter utilized in exponential phase during nitrogen limitation and a S -dependent promoter active during stationary phase. We used an ast-lacZ fusion to show that arginine stimulates expression, that ArgR, the arginine repressor, enhances expression from both promoters but is not essential, and that transcription by the two forms of the RNA polymerase is competitive and mutually exclusive. We demonstrated the binding of RNA polymerase holoenzymes, NR I , and ArgR to the promoter region in vitro. We also reconstituted transcription from both promoters with purified components, which confirmed the accessory role of ArgR for the 54 -dependent promoter. Thus, the ast operon exhibits nitrogen source-specific induction that is unique for an NR I -dependent gene. The transcriptional regulation of the ast operon in E. coli differs from that in Salmonella enterica serovar Typhimurium, in which ArgR is required for ast operon expression.

Catabolite Repression of the Propionate Catabolic Genes inEscherichia coliandSalmonella enterica: Evidence for Involvement of the Cyclic AMP Receptor Protein

“…Also, Lu and Abdelal proposed that expression of the putative 54 -dependent astC promoter in S. enterica serovar Typhimurium is subject to carbon catabolite repression and requires CRP, together with arginine and ArgR, for activation of the ast operon (23). On the other hand, despite the effect of catabolite repression on the expression of astC in E. coli, Kiupakis and Reitzer (18) were unable to demonstrate CRP binding at the astC promoter. Interestingly, unlike the 54 -dependent promoters described above, 54 -dependent prpBCDE genes are not involved in nitrogen metabolism (38).…”

Transcriptional occlusion caused by overlapping promoters

“…We denote this nitrogen-stress medium as M63 (Arg + ) in contrast to the typical M63 medium that contains the preferred nitrogen source ammonia. Arginine is catabolized via the ast pathway, and expression of these genes is induced by nitrogen stress in RpoN/NtrC-dependent fashion (25,26).…”

Phosphorylation-independent Dimer-Dimer Interactions by the Enhancer-binding Activator NtrC of Escherichia coli

“…DNase I Footprinting-Assays were performed as previously described (6,16). For binding to site 2 of the glnA regulatory region, we used a 1.6-kb EcoRI-PstI fragment from pCB37, which contains sequences from Ϫ122 to Ϫ92 (16).…”

“…For binding to site 2 of the glnA regulatory region in the presence of site 1, we used a 153-bp EcoRI-PstI fragment from pVW7, which contains sequences from Ϫ189 to Ϫ92 (16). For binding to the ast promoter, we used a 442-bp EcoRI-PstI fragment from pUC-astp(ϩ), which contains sequences from ϩ103 to Ϫ298 of the ast promoter cloned into the HincII site of pUC18 (6).…”