Isolation and analysis of mutant alleles of the Bacillus subtilis HrcA repressor with reduced dependency on GroE function

Abstract

The hrcA gene of Bacillus subtilis codes for a transcriptional repressor protein that negatively regulates expression of the heptacistronic dnaK and the bicistronic groE operon by binding to an operator-element called CIRCE. Recently, we have published data suggesting that the activity of HrcA is modulated by the GroE chaperonin system. Biochemical analyses of the HrcA protein have been hampered so far by its strong tendency to aggregate. Here, a genetic method was used to isolate mutant forms of HrcA with increased activity under conditions of decreased GroE function. One of these mutant forms (HrcA114) containing five amino acid replacements exhibited enhanced solubility when overexpressed. HrcA114 purified under native conditions produced two retarded CIRCE-containing DNA fragments in band shift experiments. The amount of the larger fragment increased after addition of GroEL, GroES, and ATP but decreased when ATP was replaced by the nonhydrolyzable ATP analog ATPγS. DNase I footprinting experiments exhibited full protection of the CIRCE element and neighboring nucleotides in an asymmetric way. An in vitro binding assay using affinity chromatography showed direct and specific interaction between HrcA114 and GroEL. All these experimental data are in full agreement with our previously published model that HrcA needs the GroE chaperonin system for activation.