Genetic dissection of Escherichia coli's master diguanylate cyclase DgcE: Role of the N-terminal MASE1 domain and direct signal input from a GTPase partner system

Abstract

The ubiquitous second messenger c-di-GMP promotes bacterial biofilm formation by playing diverse roles in the underlying regulatory networks. This is reflected in the multiplicity of diguanylate cyclases (DGC) and phosphodiesterases (PDE) that synthesize and degrade c-di-GMP, respectively, in most bacterial species. One of the 12 DGCs of Escherichia coli, DgcE, serves as the top-level trigger for extracellular matrix production during macrocolony biofilm formation. Its multi-domain architecture–a N-terminal membrane-inserted MASE1 domain followed by three PAS, a GGDEF and a degenerate EAL domain–suggested complex signal integration and transmission through DgcE. Genetic dissection of DgcE revealed activating roles for the MASE1 domain and the dimerization-proficient PAS ₃ region, whereas the inhibitory EAL ^deg domain counteracts the formation of DgcE oligomers. The MASE1 domain is directly targeted by the GTPase RdcA (YjdA), a dimer or oligomer that together with its partner protein RdcB (YjcZ) activates DgcE, probably by aligning and promoting dimerization of the PAS ₃ and GGDEF domains. This activation and RdcA/DgcE interaction depend on GTP hydrolysis by RdcA, suggesting GTP as an inhibitor and the pronounced decrease of the cellular GTP pool during entry into stationary phase, which correlates with DgcE-dependent activation of matrix production, as a possible input signal sensed by RdcA. Furthermore, DgcE exhibits rapid, continuous and processive proteolytic turnover that also depends on the relatively disordered transmembrane MASE1 domain. Overall, our study reveals a novel GTP/c-di-GMP-connecting signaling pathway through the multi-domain DGC DgcE with a dual role for the previously uncharacterized MASE1 signaling domain.

Author summary

Biofilms represent a multicellular life form of bacteria, in which large numbers of cells live in communities surrounded and protected by a self-generated extracellular polymeric matrix. As biofilms tolerate antibiotics and host immune systems, they are causally associated with chronic infections. Biofilm formation is generally promoted by the ubiquitous bacterial second messenger c-di-GMP. DgcE, one of the 12 diguanylate cyclases that produce c-di-GMP in E. coli, was previously shown to specifically act as a top level trigger in the regulatory network that drives biofilm matrix production in this bacterium. However, signal input into DgcE itself, which is a large six-domain protein, had remained unknown. Here we demonstrate that DgcE activity is controlled by a novel type of dynamin-like GTPase that directly interacts with the N-terminal membrane-intrinsic MASE1 domain of DgcE. Our finding of a dual function of this MASE1 domain, which is essential for both activation and continuous proteolysis of DgcE, is the first characterization of this widespread bacterial signaling domain. Signal input via the dynamin-like GTPase system suggests that c-di-GMP production by DgcE might be stimulated by the decreasing cellular GTP level during entry into stationary phase, which is precisely the time when biofilm matrix production is turned on.