Sites required for GltC-dependent regulation of Bacillus subtilis glutamate synthase expression

Abstract

The Bacillus subtilis gltAB genes, coding for the two subunits of glutamate synthase, are transcribed divergently from the gltC gene, encoding a LysR-type transcriptional activator of gltAB. The predicted gltA and gltC transcription start sites are separated by 51 to 52 bp. A 15-bp, consensus binding site (Box I) for LysR-type proteins was found centered at position ؊64 with respect to the gltA transcription start. This site was shown by mutational analysis to be required both for GltC-mediated activation of gltA and for autorepression of gltC. Box II, which is similar to Box I, is centered 22 bp downstream of Box I and overlaps the ؊35 region of the gltA promoter. Box II was found to be essential for activation of gltA but not for gltC autoregulation. Introduction of approximately one additional helical turn of DNA between Box I and Box II enhanced gltA expression 7-to 40-fold under nonactivating conditions and about 2-fold under activating conditions. Expression of gltA was dramatically decreased when the distance between Box I and Box II was altered by a nonintegral number of helical turns of DNA. gltC autorepression was abolished by most of the inserts between Box I and Box II but was augmented by adding one helical turn.Glutamate synthase is an important enzyme of nitrogen metabolism in bacterial cells. In Bacillus subtilis it is the enzyme responsible for biosynthesis of glutamate, an essential component of the major pathway for ammonia assimilation and a direct nitrogen donor for the biosynthesis of amino acids and other nitrogen-containing compounds (34). The reaction catalyzed by glutamate synthase, transfer of an amide group from glutamine to 2-ketoglutarate, places the enzyme at the intersection of intracellular carbon and nitrogen metabolism.In some gram-negative bacteria, expression of glutamate synthase genes is regulated positively by the leucine-responsive protein, LRP (10), or negatively by the Nac protein (3); it may also be affected by the cyclic AMP (cAMP) receptor proteincAMP complex (26). In none of these cases is the complete pattern of glutamate synthase expression understood, however.In B. subtilis, transcription of the gltA and gltB genes, encoding the large and small subunits of glutamate synthase, respectively, is subject to nitrogen-source-dependent positive regulation by the product of the gltC gene (5). GltC protein, like the Nac protein of Klebsiella aerogenes (35), belongs to the LysR family of bacterial regulators of transcription. As is the case for many members of this family (with the notable exception of Nac), GltC is encoded just upstream of and in the opposite orientation to its target genes, gltA and gltB. In addition to stimulating transcription of gltAB, GltC represses transcription of its own gene (5). Such negative autoregulation is a feature shared by many proteins of the LysR family (31).The present study was undertaken to characterize the overlapping gltA and gltC promoter regions and to identify the DNA sequences involved in regulation of these genes. MATERIALS AN...

The Arginine Regulatory Protein Mediates Repression by Arginine of the Operons Encoding Glutamate Synthase and Anabolic Glutamate Dehydrogenase in Pseudomonas aeruginosa

“…In contrast, both gltBD and gdhA of Klebsiella aerogenes are repressed by Nac (10). In B. subtilis, the gltAB operon, which encodes GOGAT, requires GltC for its activation (1) and is repressed by the global nitrogen regulator TnrA under nitrogen limitation (2,35). No Lrp or Nac functional homologues have been hitherto identified for P. aeruginosa.…”

Structure and function of the LysR-type transcriptional regulator (LTTR) family proteins

“…Broadly they bind at 235 to +20 bp (regulatory binding site, RBS, and autoregulatory site) and 240 to 220 bp (activation binding site, ABS) with hypersensitivity (associated with DNA bending) at 255 bp (Belitsky et al, 1995;Lochowska et al, 2001;Porrú a et al, 2007). However, binding sites as far away as 2218 bp with respect to the promoter region as well as internal binding sites (+350 bp; IBS), have been identified (Wilson et al, 1995;Viswanathan et al, 2007).…”

“…The gltAB operon encodes the two subunits of glutamate synthase. LTTR boxes have been identified in the promoter region of gltC and gltAB and three regions have been annotated as Box I, II and III (Belitsky et al, 1995). Box I is found at 264 bp upstream of gltA and GltC bound to this site represses expression of gltC.…”

Genome-wide identification of Bacillus subtilis CodY-binding sites at single-nucleotide resolution

“…Cells were grown in TSS medium as described (28). β-Galactosidase specific activity was determined as described previously (39). Methods for common DNA manipulations, transformation, gel shift experiments, DNase I footprinting, and sequence analysis were as previously described (12,28).…”