Thermostability, oligomerization and DNA-binding properties of the regulatory protein ArgR from the hyperthermophilic bacterium Thermotoga neapolitana

The hexameric regulatory protein ArgR formed by arginine-mediated dimerizat ion of identical trimers governs the expression of genes required for argin ine metabolism and some other genes in mesophilic and moderately thermophil ic bacteria. We have cloned the argR gene from two hyperthermophilic bacter ia of the genus Thermotoga. The two-domain ArgR proteins encoded by T. neap olitana and T. maritima share a low degree of sequence similarity with othe r bacterial arginine repressors. The ArgR protein from T. neapolitana binds to an operator located just upstream of its coding sequence and, therefore , the argR gene may be autoregulated. The protein has extremely high intrin sic thermostability and tolerance to urea. Moreover, its binding to target DNA increases the melting temperature by approximately 15 degrees C. The fo rmation of oligomeric ArgR-DNA complexes is a function of protein concentra tion, with hexameric complexes being favoured at higher concentrations. In the presence of arginine the hyperthermophilic ArgR protein binds to its ow n operator, argRo, only by forming hexamer ArgR-DNA complexes, whereas both trimer-DNA and hexamer-DNA complexes are detected in the absence of argini ne. However, the affinity of T. neapolitana ArgR for DNA has been found to be higher for a mixture of trimers and non-bound hexamers than for arginine -bound hexamers. Our data indicate that genes for arginine biosynthesis are clustered in a putative operon, which could also be regulated by the ArgR protein, in the hyperthermophilic host.