The highly thermostable arginine repressor of Bacillus stearothermophilus: gene cloning and repressor–operator interactions

Abstract

SummaryWe report here the cloning of the arginine repressor gene argR of Bacillus stearothermophilus and the characterization and purification to homogeneity of its product. The deduced amino acid sequence of the 16.8-kDa ArgR subunit shares 72% identity with its mesophilic homologue AhrC of Bacilus subtilis. Sequence analysis of B. stearothermophilus ArgR and comparisons with mesophilic arginine repressors suggest that the thermostable repressor comprises an N-terminal DNA-binding and a C-terminal oligomerization and arginine-binding region. B. stearothermophilus ArgR has been overexpressed in E. coli and purified as a 48.0-kDa trimeric protein. The repressor inhibits the expression of a B. stearothermophilus argC-lacZ fusion in E. coli cells. In the presence of arginine, the purified protein binds tightly and specifically to the argC operator, which largely overlaps the argC promoter. The purified B. stearothermophilus repressor proved to be very thermostable with a half-life of approximately 30 min at 90ЊC, whereas B. subtilis AhrC was largely inactivated at 65ЊC. Moreover, ArgR operator complexes were found to be remarkably thermostable and could be formed efficiently at up to 85ЊC, well above the optimal growth temperature of the moderate thermophile B. stearothermophilus. This pronounced resistance of the repressor-operator complexes to heat treatment suggests that the same type of regulatory mechanism could operate in extreme thermophiles.

The structure of AhrC, the arginine repressor/activator protein fromBacillus subtilis

“…In the absence of l-arginine, hexamers are in rapid equilibrium with trimers (Holtham et al, 1999). The position of equilibrium seems to differ for different homologues, with trimers being dominant for B. stearothermophilus ArgR (ArgRBst; Dion et al, 1997;Ni et al, 1999), but hexamers favoured for AhrC (Czaplewski et al, 1992), ArgREc (Lim et al, 1987) and S. typhimurium ArgR (Lu et al, 1992).…”

“…Arginine-regulatory proteins, which are usually called ArgR in organisms other than B. subtilis, have been identi®ed in E. coli (Lim et al, 1987), B. stearothermophilus (Dion et al, 1997) and Salmonella typhimurium (Lu et al, 1992). These proteins have been biochemically characterized and shown to act as repressors of arginine biosynthesis in their respective hosts, although any roles in the activation of arginine catabolism remain to be con®rmed experimentally.…”

“…These proteins have been biochemically characterized and shown to act as repressors of arginine biosynthesis in their respective hosts, although any roles in the activation of arginine catabolism remain to be con®rmed experimentally. Sequences are also known for probable AhrC/ArgR homologues from Clostridium perfringens (Ohtani et al, 1997), Haemophilus in¯uenzae (Fleischmann et al, 1995), Mycobacterium tuberculosis (Cole et al, 1998), Streptomyces clavuligerus (Rodriguez-Garcia et al, 1997) and Streptococcus pneumoniae (Priebe et al, 1988).…”

Interaction between ArgR and AhrC Controls Regulation of Arginine Metabolism in Lactococcus lactis

“…Despite differences in the organization of genes involved in arginine metabolism, experimental evidence indicates that the mechanism of arginine-dependent regulation of these genes is highly conserved among a range of different organisms, including Gram-negative, Gram-positive and extremophilic bacteria (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). Regulation is exerted by binding of single transcriptional regulators of the ArgR family to so-called ARG operator sites preceding the relevant target genes, generally leading to repression of arginine biosynthetic genes and activation of catabolic genes, in the presence of arginine.…”

“…The subunits are arranged in hexameric structures, which are organized as dimers of trimers. In E. coli and B. subtilis, the hexameric structure is maintained both in the absence and presence of arginine (4,17), whereas the regulator of B. stearothermophilus mainly exists as a trimer that assembles into hexamers dependent of the concentrations of arginine, protein, and DNA (5,15). Six arginine molecules are bound at the trimer-trimer interface, strengthening the interaction between the trimers and at the same time introducing a conformational change in the regulator, thus increasing its affinity for operator binding (4).…”