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...
