Hyperthermophilic Thermotoga Arginine Repressor Binding to Full-length Cognate and Heterologous Arginine Operators and to Half-site Targets

Abstract

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 (ArgRTn) 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 ArgRTn binding to the operator of the biosynthetic argGHCJBD operon of Thermotoga maritima indicates that ArgRTn 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 ArgRTn 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 Kd of ArgRTn 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 ArgRTn, instead of the highly conserved glutamine that is involved in arginine binding in the E.coli repressor. Consistent with these results, ArRTn 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 ArgRTn, owing to its broad target specificity and its ability to bind single ARG box sequences, might play a more general regulatory role in Thermotoga