Structural and biochemical investigations into c-di-GMP signaling in "E.coli" : Zinc dependent regulation of the diguanylate cyclase YdeH and characterization of PgaA and PgaB, involved in c-di-GMP controlled exopolysaccharide synthesis

In response to adverse conditions, many bacterial species can switch from a planktonic growth to a surface associated growth mode and form biofilm communities. A key factor triggering the formation of biofilms in a multitude of bacterial species is the second messenger bis-(3’-5’)cyclic dimeric guanosine (c-di-GMP). The biosynthesis of c-di-GMP by condensation of two GTP molecules is performed by diguanylate cyclases (DGCs). DGCs consist of catalytic GGDEF domains in combination with N-terminal, environment sensing regulatory domains. A significant fraction of DGCs are linked to N-terminal sequences of unknown function indicating that c-di-GMP signaling is linked to numerous undiscovered environmental and cellular signals. In this study structural and biochemical analysis on the DGC YdeH from E. coli was undertaken, to elucidate its regulatory mechanism. Three-dimensional structures of YdeH were determined, which reveal in the regulation of YdeH. The N-terminal sensory domain of YdeH shows a new fold, a four helical bundle, which harbors a zinc-binding site compromised of three histidines and one cysteine. It could be shown that the DGC activity of YdeH is inhibited by zinc binding to the N- terminal sensory domain with an inhibition constant in the femtomolar range. A model for the inhibition of YdeH by zinc is proposed, in which upon zinc binding the linker between the regulatory domain and the enzymatic domain is fixed in a conformation, which prevents the productive encounter of the two GGDEF domains.
In the structures of YdeH, substrate and product binding to the active site could be shown, however the dimeric arrangement of the two DGC domains, each harboring only one half of the active site, are not in a competent constellation. With the help of the determined structures of YdeH a model of a competent dimer was generated, which provides insights into the regulation of YdeH. Product binding to the inhibitory site of YdeH was shown in the crystal structures and inhibition by c-di-GMP was demonstrated in enzymatic experiments. YdeH represents the first example of a biological zinc-sensor that exerts its downstream effects post-transcriptionally and the first example of a metal sensory c-di-GMP signaling protein.
A protocol for the enzymatic large-scale synthesis of c-di-GMP by using the DGC YdeH from E. coli was developed and optimized. In contrast to the chemical synthesis of c-di- GMP, enzymatic c-di-GMP production is a one-step reaction that can easily be performed with the equipment of a standard biochemical lab. The protocol allows the production of milligram amounts of c-di-GMP within one day and paves the way for extensive biochemical and biophysical studies on c-di-GMP-mediated processes.
In biofilms cells are entrapped within a extracellular polymeric matrix. One component of this matrix is the poly-β-1,6-N-Acetyl-glucosamine (poly-1,6-GlcNAc), which is synthesized and exported by the four proteins of the pgaABCD operon. PgaC and PgaD are responsible for the synthesis of poly-1,6-GlcNAc and are allosterically regulated by c-di-GMP. The deacetylase PgaB and the outer membrane protein PgaA are involved in the modification and export of the poly-1,6-GlcNAc chain.
For PgaA and PgaB an expression and purification protocol was established and resulted in stable and homogenous proteins. The predicted deacetylase activity of PgaB was demonstrated in vitro with an activity assay, which is suitable for rapid screening of different reaction conditions and for the search of inhibitors for PgaB and PgaC, which are of specific pharmaceutical interest.