Negative Transcriptional Regulation of the ilv-leu Operon for Biosynthesis of Branched-Chain Amino Acids through the Bacillus subtilis Global Regulator TnrA

Abstract

The Bacillus subtilis ilv-leu operon is involved in the synthesis of branched-chain amino acids (valine, isoleucine, and leucine). The two-to threefold repression of expression of the ilv-leu operon during logarithmicphase growth under nitrogen-limited conditions, which was originally detected by a DNA microarray analysis to compare the transcriptomes from the wild-type and tnrA mutant strains, was confirmed by lacZ fusion and Northern experiments. A genome-wide TnrA box search revealed a candidate box approximately 200 bp upstream of the transcription initiation base of the ilv-leu operon, the TnrA binding to which was verified by gel retardation and DNase I footprinting analyses. Deletion and base substitution of the TnrA box sequence affected the ilv-leu promoter activity in vivo, implying that TnrA bound to the box might be able to inhibit the promoter activity, possibly through DNA bending. The negative control of the expression of the ilv-leu operon by TnrA, which is considered to represent rather fine-tuning (two-to threefold), is a novel regulatory link between nitrogen and amino acid metabolism.Branched-chain amino acids (isoleucine, valine, and leucine) are the most abundant amino acids in proteins and form the hydrophobic core of proteins. In addition, these amino acids are the precursors for the biosynthesis of iso-and anteisobranched fatty acids, which represent the major fatty acid species of the membrane lipids in Bacillus species (3). The initial step in isoleucine and valine synthesis is the condensation of pyruvate and threonine and two pyruvates, respectively, leading to the formation of branched-chain keto acids (5). Leucine is synthesized from one of the branched-chain keto acids, ␣-ketoisovalerate.The ilv-leu operon of Bacillus subtilis comprises seven genes necessary for the biosynthesis of isoleucine, valine, and leucine ( Fig. 1). Previous studies demonstrated that the ilv-leu operon is regulated in response to leucine availability by the T-box transcription antitermination system (8, 9, 15). The common T-box-dependent regulatory mechanism for the ilv-leu operon and the aminoacyl-tRNA synthetase genes (10, 18) could result in the coregulation of these genes. However, regulation of the ilv-leu operon solely by leucine availability would create a potential problem for the cell, because excess leucine could cause the cell to be starved of isoleucine and valine, which are also the end products of the isoleucine-leucine biosynthetic pathway encoded by ilv-leu.Recently, global studies on B. subtilis gene expression in response to amino acid availability and subsequent analysis of the expression of the genes for branched-chain amino acid biosynthesis (13,14,17) revealed that the ilv-leu operon is downregulated in the presence of the 16 amino acids included in casein, which is independent of the T-box transcription antitermination system but dependent on negative regulation through CodY. Very recently, Shivers and Sonenshein (21) demonstrated that CodY is activated by direct interaction with bra...

Cross-Regulation of the Bacillus subtilis glnRA and tnrA Genes Provides Evidence for DNA Binding Site Discrimination by GlnR and TnrA

“…sion of the ilv-leu operon is also inhibited by the binding of TnrA to a site located 200 bp upstream of the transcriptional start site (38). The molecular mechanism for this mode of transcription inhibition of these two promoters is not known.…”

Carbon Catabolite Control of the Metabolic Network inBacillus subtilis

“…119) Besides the probable necessity that the CcpA-dependent CCA of ilv-leu in glycolysis proceed continuously for the draining of accumulated pyruvate, it is notable that this positive regulation links carbon metabolism to amino acid anabolism. Recent global gene expression studies on amino acid availability 120) and CodY regulation, 121) as well as on the metabolic links of ilv-leu expression to glucose and nitrogen metabolism, 38,39,98,122) indicate that the ilv-leu operon is under direct negative transcriptional control through two major global regulators of nitrogen metabolism (CodY and TnrA).…”

CcpA-Mediated Catabolite Activation of the Bacillus subtilis ilv-leu Operon and Its Negation by Either CodY- or TnrA-Mediated Negative Regulation

“…This CA of the ilv-leu operon is also negated by TnrA through its binding to the TnrA box located at nt Ϫ209/Ϫ193 (Fig. 1) (not nt Ϫ211/Ϫ195, as incorrectly described in reference 10) in cells grown under nitrogen-limited conditions (10). It was assumed that this TnrA-mediated negative regulation to negate CA might occur through an interaction of TnrA bound to the TnrA box with the complex of CcpA and P-Ser-HPr bound to the cre site (6).…”

“…The CcpA-mediated CA of ilv-leu is also negatively regulated by TnrA through its binding to the TnrA box located at nt Ϫ209/Ϫ193 under nitrogen-limited conditions (10). The target genes and operons of the TnrA protein were roughly estimated at 25, including ilv-leu (10,40) (40).…”

“…1), links carbon metabolism to AA anabolism. Global gene expression studies on AA availability (8) and CodY regulation (9), as well as a study on metabolic linking of ilv-leu expression to nitrogen metabolism (10), revealed that the ilv-leu operon is under direct negative transcriptional control through two major global regulators of nitrogen metabolism (TnrA and CodY) (Fig. 1).…”