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 PAS3 region, whereas the inhibitory EALdeg 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 PAS3 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.
Highlights
The discovery and functional analysis of bacterial cyclic dinucleotides, in particular bis-(3 ́,5 ́)cyclic diguanosine monophosphate (c-di-GMP), has opened a new era in the study of second messenger signaling in prokaryotes, revealing a complexity that matches that in eukaryotes [1,2,3,4]
What is the role of all these domains in signal integration and transduction into and through DgcE? Can an analysis of DgcE lead us to an understanding of the sensory function of the widespread membrane-integral MASE1 domain? Are any other factors involved in signal input into DgcE? To elucidate the molecular functions of DgcE, we focussed on a detailed genetic analysis since DgcE is a complex membrane-associated protein, but in the course of our experiments we discovered a constitutive in-vivo turnover of DgcE, i.e. DgcE has properties that efficiently prevent purification and in-vitro analysis
Role of the MASE1 domain and GTPase control of the master diguanylate cyclase DgcE of E. coli deletion of dgcE (Fig 1C), i.e. a phenotype corresponding to a strong reduction in both curli fibres and pEtN-cellulose [13]
Summary
The discovery and functional analysis of bacterial cyclic dinucleotides, in particular bis-(3 ́,5 ́)cyclic diguanosine monophosphate (c-di-GMP), has opened a new era in the study of second messenger signaling in prokaryotes, revealing a complexity that matches that in eukaryotes [1,2,3,4]. C-di-GMP is synthesized by diguanylate cyclases (DGCs, with this activity provided by their GGDEF domains) and degraded by specific phosphodiesterases (PDEs, with either EAL or HD-GYP domains) [5, 6] These signaling enzymes usually exhibit a modular domain architecture. Diverse N-terminal domains, which in many cases anchor the enzymes in the cytoplasmic membrane, act as signal input devices responding to often still unknown signals These DGCs and PDEs antagonistically negotiate levels of c-diGMP which is bound by a large diversity of effector components. These can be different types of proteins that control various targets by direct interaction or riboswitches in the upstream regions of mRNAs that affect transcriptional elongation or translation [7, 8]. Local c-di-GMP signaling has been observed, especially in bacterial species that have multiple DGCs and PDEs, some of which can exert surprisingly specific functions based on their direct association with effector/target systems [9,10,11,12,13,14]. c-di-GMPcontrolled targets are involved in fundamentally important cellular and physiological functions, such as the formation of highly antibiotic-tolerant biofilms, motility, virulence, cell cycle progression and development [3, 15,16,17,18,19]
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