DNA-binding properties of a cGMP-binding CRP homologue that controls development of metabolically dormant cysts of Rhodospirillum centenum

摘要

Rhodospirillum centenum utilizes 3′,5′-cyclic guanosine monophosphate (cGMP) as a messenger to regulate development of desiccation-resistant cysts. In this study, we demonstrated that gcyA, gcyB and gcyC, coding for putative subunits of a guanylyl cyclase, increase expression from 8- to 500-fold when cells transition from vegetative to cyst phases of growth. This induction did not occur in a strain that is defective in cGMP synthesis or in a strain that contains a deletion of cgrA that codes for a cGMP-binding homologue of Escherichia coli catabolite repressor protein (CRP). We also demonstrated that cgrA auto-induces its own expression in the presence of cGMP, indicating that a feed-forward loop is used to ramp up cGMP production as cells undergo encystment. Inspection of an intragenic region upstream of gcyB revealed a sequence that is identical to the CRP consensus sequence from E. coli. DNase I and fluorescence anisotropy analyses demonstrated that CgrA bound to this target sequence at a protein : cGMP ratio of 1 : 2 with Kd ∼61 nM. This was in contrast to CgrA in the presence of cAMP, which exhibited Kd ∼1795 nM. CgrA thus constitutes a novel variant of CRP that utilizes cGMP to regulate production of cGMP synthase for the control of cyst development.