SummaryThe Bacillus subtilis ilv-leu operon involved in the biosynthesis of branched-chain amino acids is under negative regulation mediated by TnrA and CodY, which recognize and bind to their respective cis -elements located upstream of the ilv-leu promoter. This operon is known to be under CcpA-dependent positive regulation. We have currently identified a cataboliteresponsive element (
Branched-chain amino acids are the most abundant amino acids in proteins. The Bacillus subtilis ilv-leu operon is involved in the biosynthesis of branched-chain amino acids. This operon exhibits a RelA-dependent positive stringent response to amino acid starvation. We investigated this positive stringent response upon lysine starvation as well as decoyinine treatment. Deletion analysis involving various lacZ fusions revealed two molecular mechanisms underlying the positive stringent response of ilv-leu, i.e., CodY-dependent and -independent mechanisms. The former is most likely triggered by the decrease in the in vivo concentration of GTP upon lysine starvation, GTP being a corepressor of the CodY protein. So, the GTP decrease derepressed ilv-leu expression through detachment of the CodY protein from its cis elements upstream of the ilv-leu promoter. By means of base substitution and in vitro transcription analyses, the latter (CodY-independent) mechanism was found to comprise the modulation of the transcription initiation frequency, which likely depends on fluctuation of the in vivo RNA polymerase substrate concentrations after stringent treatment, and to involve at least the base species of adenine at the 5 end of the ilv-leu transcript. As discussed, this mechanism is presumably distinct from that for B. subtilis rrn operons, which involves changes in the in vivo concentration of the initiating GTP.Branched-chain amino acids are the most abundant amino acids in proteins and form the hydrophobic cores of the proteins. Moreover, these amino acids are precursors for the biosynthesis of iso-and anteiso-branched fatty acids, which represent the major fatty acid species of the membrane lipids in Bacillus species (5). The initial step of isoleucine or valine synthesis is the condensation of 2-oxobutanoate derived from threonine and pyruvate or two pyruvates, leading to the formation of branched-chain keto acids (8). Leucine is synthesized from one of the branched-chain keto acids, i.e., ␣-ketoisovalerate. The Bacillus subtilis ilv-leu operon comprises seven genes (ilvBHC and leuABCD) necessary for the biosynthesis of branched-chain amino acids (12). The expression of the ilv-leu operon is under positive regulation involving the CcpA protein (36, 41), which is involved in carbon catabolite control of not only hundreds of the catabolic operons and genes but also many cellular processes (6, 11). This CcpAdependent positive regulation of ilv-leu links carbon metabolism to amino acid anabolism. Recent global gene expression studies of amino acid availability (23) and CodY regulation (25), as well as studies of metabolic linking of ilv-leu expression to nitrogen metabolism (40), revealed that the ilv-leu operon is under direct negative transcriptional control through two major global regulators of nitrogen metabolism (TnrA and CodY). TnrA is known to both activate and repress nitrogenregulated genes during nitrogen-limited growth (43). The CodY protein is a GTP-binding repressor of several operons, including ilv-leu, that...
A gene encoding an ADP-dependent phosphofructokinase homologue has been identified in the hyperthermophilic archaeon Methanococcus jannaschii via genome sequencing. The gene encoded a protein of 462 amino acids with a molecular weight of 53,361. The deduced amino acid sequence of the gene showed 52 and 29% identities to the ADP-dependent phosphofructokinase and glucokinase from Pyrococcus furiosus, respectively. The gene was overexpressed in Escherichia coli, and the produced enzyme was purified and characterized. To our surprise, the enzyme showed high ADP-dependent activities for both glucokinase and phosphofructokinase. A native molecular mass was estimated to be 55 kDa, and this indicates the enzyme is monomeric. The reaction rate for the phosphorylation of D-glucose was almost 3 times that for D-fructose 6-phosphate. The K m values for D-fructose 6-phosphate and D-glucose were calculated to be 0.010 and 1.6 mM, respectively. The K m values for ADP were 0.032 and 0.63 mM when D-glucose and D-fructose 6-phosphate were used as a phosphoryl group acceptor, respectively. The gene encoding the enzyme is proposed to be an ancestral gene of an ADP-dependent phosphofructokinase and glucokinase. A gene duplication event might lead to the two enzymatic activities.In general, ATP is regarded as the universal energy carrier and the most common phosphoryl group donor for kinases. However, several gluco-and phosphofructokinases have been reported to have different phosphoryl group donor specificity. The glucokinase from Mycobacterium tuberculosis can utilize both ATP and polyphosphate as the phosphoryl group donor (1). PP i -dependent phosphofructokinases have been reported to be present in several eucarya and bacteria and in the hyperthermophilic archaeon Thermoproteus tenax (2-4). Recently novel sugar kinases, ADP-dependent (AMP-forming) glucokinase (ADP-GK) 1 and phosphofructokinase (ADP-PFK), were discovered in the hyperthermophilic archaeon Pyrococcus furiosus (5). Those enzymes require ADP as the phosphoryl group donor instead of ATP and are involved in a modified EmbdenMeyerhof pathway in this organism. The hyperthermophilic archaea are relatively deeply branched archaea and are considered to be phylogenetically ancient organisms. Therefore, structural analysis of the kinases from these organisms may provide abundant information for phylogenetic analysis of the sugar kinases. We cloned and sequenced the gene encoding the ADP-GKs from P. furiosus and Thermococcus litoralis (The nucleotide sequences have been submitted to the GenBank TM data bases as the genes for ADP-dependent hexokinase and are available under accession numbers E14588 and E14589.) (6). About 59% identity in amino acid sequence was observed between these two enzymes, although they did not show similarity with any ATP-dependent kinases that have been reported so far. In addition, the amino acid sequence of the P. furiosus ADP-GK showed high identity (26%) with that reported for the P. furiosus ADP-PFK (7). This suggests that those kinases belong to a...
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...
During DNA microarray analysis, we discovered that the GlnK-GlnL (formerly YcbA-YcbB) two-component system positively regulates the expression of the glsA-glnT (formerly ybgJ-ybgH) operon in response to glutamine in the culture medium on Northern analysis. As a result of gel retardation and DNase I footprinting analyses, we found that the GlnL protein interacts with a region (bases ؊13 to ؊56; ؉1 is the transcription initiation base determined on primer extension analysis of glsA-glnT) in which a direct repeat, TTTTGTN4T TTTGT, is present. Furthermore, the glsA and glnT genes were biochemically verified to encode glutaminase and glutamine transporter, respectively.Various organisms have developed sophisticated signaling systems for eliciting a variety of adaptive responses to their environment. The two-component regulatory system is one such signaling system in prokaryotes, lower eukaryotes, and plants, and it consists of at least two signal transduction proteins (the sensor kinase and the response regulator). The sensor kinase senses environmental or intercellular signals and then transmits them to the response regulator through phosphoryl group transfer. The response regulator often binds to the promoter regions of target genes and regulates their transcription through activation or repression (28).Genome sequencing of Bacillus subtilis has revealed the presence of 36 sensor kinases and 35 response regulators, each of 30 kinase-regulator pairs residing in an operon on the genome (20). According to the classification of sensor kinases as to the sequence around the phosphorylated histidine, CitS, DctS, MalK, and GlnK (formerly YcbA) belong to the same group of kinases (group IV), which are paired with CitT, DctT, MalR, and GlnL (formerly YcbB) on the genome, respectively (9). Out of the four kinase-regulator pairs, three (the exception being GlnKL) have been reported to sense the presence of the respective tricarboxylic acid cycle intermediates in the culture medium and to positively regulate each system of their utilization. The CitST system positively regulates the expression of citM, which encodes the Mg-citrate transporter (36), whereas DctST enhances the transcription of the ydbEFGH operon, which is involved in the utilization of fumarate and succinate (3). Recently, the MalKR system was reported to trigger the transcription of ywkA, which encodes a malic enzyme (8), and that of maeN and yflS (30), which encodes malate transporters, indicating that this system is involved in the transport and utilization of malate. Thus, only the function of the fourth system (GlnKL) belonging to this group remained to be characterized.Glutamine, the best nitrogen source for B. subtilis, serves as an amino acid for protein synthesis and a nitrogen donor for the synthesis of various nitrogenous molecules in the cell (10). It also functions as a gauge of the nitrogen supply level in the cell, being a feedback inhibitor of glutamine synthetase. Glutamine synthetase interacting with glutamine is able to trap and inhibit TnrA, w...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2025 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.