DNA binding of Escherichia coli arginine repressor mutants altered in oligomeric state

The Escherichia coli arginine repressor (ArgR) controls expression of the arginine biosynthetic genes and acts as an accessory protein in Xer site-specific recombination at cer and related plasmid recombination sites. The hexameric wild-type protein shows L -arginine-dependent DNA binding. In this work, ArgR mutants that are defective in trimer–trimer interactions and bind DNA as trimers in an L -arginine-independent manner are isolated and characterized. Whereas the wild-type ArgR hexamer exhibits high-affinity binding to two repeated ARG boxes separated by 3 bp (each ARG box containing two identical dyad symmetrical 9 bp half-sites), the trimeric mutants bind to and footprint three adjacent half-sites of this 'idealized' substrate. Trimeric ArgR is impaired in its ability to repress the arginine biosynthetic genes and in Xer site-specific recombination. In the absence of L -arginine, residual wild-type ArgR-binding occurs as trimers. The binding of an N-terminal 77-amino-acid DNA-binding domain to idealized ARG boxes is also characterized.     

Regulation of arginine biosynthesis in the psychropiezophilic bacterium Moritella profunda: in vivo repressibility and in vitro repressor-operator contact probing

We report the cloning of the arginine repressor gene from the psychropiezophilic Gram-negative bacterium Moritella profunda, the purification of its product (ArgR(Mp)), the identification of the operator in the bipolar argECBFGH(A) operon, in vivo repressibility studies, and an in vitro analysis of the repressor-operator interaction, including binding to mutant and heterologous arginine operators. The ArgR(Mp) subunit shows about 70% amino acid sequence identity with Escherichia coli ArgR (ArgR(Ec)). Binding of purified hexameric ArgR(Mp) to the control region of the divergent operon proved to be arginine-dependent, sequence-specific, and significantly more sensitive to heat than complex formation with ArgR(Ec). ArgR(Mp) binds E.coli arginine operators very efficiently, but hardly recognizes the operator from Bacillus stearothermophilus or Thermotoga maritima. ArgR(Mp) binds to a single site overlapping the -35 element of argC(P), but not argE(P). Therefore, the arrangement of promoter and operator sites in the bipolar argECBFGH(A) operon of M.profunda is very different from the organization of control elements in the bipolar argECBH operon of E.coli, where both promoters overlap the common operator and are equally repressible. We demonstrate that M.profunda argC(P) is about 44-fold repressible, whereas argE(P) is fully constitutive. A high-resolution contact map of the ArgR(Mp)-operator interaction was established by enzymatic and chemical footprinting, missing contact and base-specific premodification binding interference studies. The results indicate that the argC operator consists of two ARG box-like sequences (18bp imperfect palindromes) separated by 3bp. ArgR(Mp) binds to one face of the DNA helix and establishes contacts with two major groove segments and the intervening minor groove of each ARG box, whereas the minor groove segment facing the repressor at the center of the operator remains largely uncontacted. This pattern is reminiscent of complex formation with the repressors of E.coli and B.stearothermophilus, and suggests that each ARG box is contacted by two ArgR subunits belonging to opposite trimers. Moreover, the premodification interference patterns and mutant studies clearly indicate that the inner, center proximal halves of each ARG box in the M.profunda argC operator are more important for complex formation and repression than the outermost halves. A close inspection of sequence conservation and of single base-pair O(c)-type mutations indicate that the same conclusion can be generalized to E.coli operators.     

Hyperthermophilic Thermotoga arginine repressor binding to full-length cognate and heterologous arginine operators and to half-site targets

The degree of sequence conservation of arginine repressor proteins (ArgR) and of the cognate operators (tandem pairs of 18 bp imperfect palindromes, ARG boxes) in evolutionarily distant bacteria is unusually high, and the global mechanism of ArgR-mediated regulation appears to be similar. However, here we demonstrate that the arginine repressor from the hyperthermophilic bacterium Thermotoga neapolitana (ArgR(Tn)) exhibits characteristics that clearly distinguish this regulator from the well-studied homologues from Escherichia coli, Bacillus subtilis and B.stearothermophilus. A high-resolution contact map of ArgR(Tn) binding to the operator of the biosynthetic argGHCJBD operon of Thermotoga maritima indicates that ArgR(Tn) establishes all of its strong contacts with a single ARG box-like sequence of the operator only. Protein array and electrophoretic mobility-shift data demonstrate that ArgR(Tn) has a remarkable capacity to bind to arginine operators from Gram-negative and Gram-positive bacteria, and to single ARG box-bearing targets. Moreover, the overall effect of L-arginine on the apparent K(d) of ArgR(Tn) binding to various cognate and heterologous operator fragments was minor with respect to that observed with diverse bacterial arginine repressors. We demonstrate that this unusual behaviour for an ArgR protein can, to a large extent, be ascribed to the presence of a serine residue at position 107 of ArgR(Tn), instead of the highly conserved glutamine that is involved in arginine binding in the E.coli repressor. Consistent with these results, ArR(Tn) was found to behave as a superrepressor in E.coli, inhibiting growth in minimal medium, even supplemented with arginine, whereas similar constructs bearing the S107Q mutant allele did not inhibit growth. We assume that ArgR(Tn), owing to its broad target specificity and its ability to bind single ARG box sequences, might play a more general regulatory role in Thermotoga     

Crystal structure of the arginine repressor protein in complex with the DNA operator from Mycobacterium tuberculosis

The arginine repressor (ArgR) from Mycobacterium tuberculosis (Mtb) is a gene product encoded by the open reading frame Rv1657. It regulates the l-arginine concentration in cells by interacting with ARG boxes in the promoter regions of the arginine biosynthesis and catabolism operons. Here we present a 2.5-Å structure of MtbArgR in complex with a 16-bp DNA operator in the absence of arginine. A biological trimer of the protein-DNA complex is formed via the crystallographic 3-fold symmetry axis. The N-terminal domain of MtbArgR has a winged helix-turn-helix motif that binds to the major groove of the DNA. This structure shows that, in the absence of arginine, the ArgR trimer can bind three ARG box half-sites. It also reveals the structure of the whole MtbArgR molecule itself containing both N-terminal and C-terminal domains.     

Two arginine repressors regulate arginine biosynthesis in Lactobacillus plantarum

The repression of the carAB operon encoding carbamoyl phosphate synthase leads to Lactobacillus plantarum FB331 growth inhibition in the presence of arginine. This phenotype was used in a positive screening to select spontaneous mutants deregulated in the arginine biosynthesis pathway. Fourteen mutants were genetically characterized for constitutive arginine production. Mutations were located either in one of the arginine repressor genes (argR1 or argR2) present in L. plantarum or in a putative ARG operator in the intergenic region of the bipolar carAB-argCJBDF operons involved in arginine biosynthesis. Although the presence of two ArgR regulators is commonly found in gram-positive bacteria, only single arginine repressors have so far been well studied in Escherichia coli or Bacillus subtilis. In L. plantarum, arginine repression was abolished when ArgR1 or ArgR2 was mutated in the DNA binding domain, or in the oligomerization domain or when an A123D mutation occurred in ArgR1. A123, equivalent to the conserved residue A124 in E. coli ArgR involved in arginine binding, was different in the wild-type ArgR2. Thus, corepressor binding sites may be different in ArgR1 and ArgR2, which have only 35% identical residues. Other mutants harbored wild-type argR genes, and 20 mutants have lost their ability to grow in normal air without carbon dioxide enrichment; this revealed a link between arginine biosynthesis and a still-unknown CO2-dependent metabolic pathway. In many gram-positive bacteria, the expression and interaction of different ArgR-like proteins may imply a complex regulatory network in response to environmental stimuli.