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PleD from Caulobacter crescentus, a response regulator with a diguanylate cyclase (DGC) domain.

BBa_K2012002 pleD

Intracellular c-di-GMP concentration has been regulated by two functionally opposing enzymes, the diguanylate cyclases (DGCs) containing a GGDEF domain, and phosphodiesterases (PDEs) containing either an EAL or HD-GYP domain.

PleD from Caulobacter crescentus, a response regulator with a diguanylate cyclase (DGC) domain, in its activated form.

Mechanistic Model of PleD Regulation(Wassmann, Chan et al. 2007)

The DGC domain (green) is connected via a flexible linker to the stem (receiver domain D1 [red] and adaptor domain D2 [yellow]) and is supposed to be mobile relative to it. (Upper row) Activation. Phosphorylation of domain D1 leads to a rearrangement of the stem domains, which, in turn, allows for formation of a tight dimeric stem (3). The dimeric arrangement is a prerequisite for an efficient and productive encounter of the two substrate-loaded DGC domains to form the c-di-GMP product (4). (Lower row) Product inhibition. Dimeric product molecules, (c-di-GMP)2, can crosslink the primary inhibition site on DGC, Ip, with a secondary binding site either on D2, Is,D2 (5) or on the adjacent DGC domain, Is,DGC (6). The DGC domains become immobilized, and the active sites are hampered from a productive encounter. Note that a possible direct communication between Ip and A sites is not depicted.

Structure and physiological functions of c-di-GMP (Hengge 2009)

At the cellular level, bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) is controlled by diguanylate cyclases that carry GGDEF domains (red) and specific phosphodiesterases that carry EAL or HD-GYP domains (blue). c-di-GMP can reduce motility by downregulating flagellar expression (for example, in Pseudomonas aeruginosa) or assembly (for example, in Caulobacter crescentus) or interfering with flagellar motor function (for example, in Escherichia coli and C. crescentus; for a review). Low c-di-GMP levels are required for the expression of acute virulence genes (for example, in Vibrio cholerae). In all bacteria tested, high c-di-GMP levels stimulated various biofilm-associated functions, such as the formation of fimbriae and other adhesins and various matrix exopolysaccharides4,6. In C. crescentus, precisely timed and localized action of c-di-GMP is a key step in cell cycle progression13. pGpG, 5’-phosphoguanylyl-(3′-5′)- guanosine.

Congo red staining

For demonstrating expression of pleD, we used Congo red staining assay. As previous mentioned, high concentration of c-di-GMP could induce E. coli synthesize exopolysaccharides, and Congo red binding is a complex phenotype that reflects various outer membrane and surface properties including the presence of adhesive structures such as curli fimbria which are involved in biofilm formation. Wild type: DE3 contain pET28b plasmid, colony which was stained red color contain pET-pleD plasmid.

Reference:

Hengge, R. (2009). "Principles of c-di-GMP signalling in bacteria." Nat Rev Microbiol 7(4): 263-273.

Wassmann, P., et al. (2007). "Structure of BeF3- -modified response regulator PleD: implications for diguanylate cyclase activation, catalysis, and feedback inhibition." Structure 15(8): 915-927.