Catabolite repression of the Bacillus subtilis gnt operon exerted by two catabolite-responsive elements

Catabolite repression of Bacillus subtilis catabolic operons is supposed to occur via a negative regulatory mechanism involving the recognition of a cis-acting catabolite-responsive element (cre) by a complex of CcpA, which is a member of the GalR-Lacl family of bacterial regulatory proteins, and the seryl-phosphorylated form of HPr (P-ser-HPr), as verified by recent studies on catabolite repression of the gnt operon. Analysis of the gnt promoter region by deletions and point mutations revealed that in addition to the cre in the first gene (gntR) of the gnt operon (cre(down)), this operon contains another cre located in the promoter region (cre(up)). A translational gntR'-'lacZ fusion expressed under the control of various combinations of wild-type and mutant cre(down) and cre(up) was integrated into the chromosomal amyE locus, and then catabolite repression of beta-galactosidase synthesis in the resultant integrants was examined. The in vivo results implied that catabolite repression exerted by cre(up) was probably independent of catabolite repression exerted by cre(down); both cre(up) and cre(down) catabolite repression involved CcpA. Catabolite repression exerted by cre(up) was independent of P-ser-HPr, and catabolite repression exerted by cre(down), was partially independent of P-ser-HPr. DNase I footprinting experiments indicated that a complex of CcpA and P-ser-HPr did not recognize cre(up), in contrast to its specific recognition of cre(down). However, CcpA complexed with glucose-6phosphate specifically recognized cre(up) as well as cre(down), but the physiological significance of this complexing is unknown.