Trigger phosphodiesterases as a novel class of c-di-GMP effector proteins

Abstract

The bacterial second messenger c-di-GMP controls bacterial biofilm formation, motility, cell cycle progression, development and virulence. It is synthesized by diguanylate cyclases (with GGDEF domains), degraded by specific phosphodiesterases (PDEs, with EAL of HD-GYP domains) and sensed by a wide variety of c-di-GMP-binding effectors that control diverse targets. c-di-GMP-binding effectors can be riboswitches as well as proteins with highly diverse structures and functions. The latter include 'degenerate' GGDEF/EAL domain proteins that are enzymatically inactive but still able to bind c-di-GMP. Surprisingly, two enzymatically active 'trigger PDEs', the Escherichia coli proteins PdeR and PdeL, have recently been added to this list of c-di-GMP-sensing effectors. Mechanistically, trigger PDEs are multifunctional. They directly and specifically interact with a macromolecular target (e.g. with a transcription factor or directly with a promoter region), whose activity they control by their binding and degradation of c-di-GMP-their PDE activity thus represents the c-di-GMP sensor or effector function. In this process, c-di-GMP serves as a regulatory ligand, but in contrast to classical allosteric control, this ligand is also degraded. The resulting kinetics and circuitry of control are ideally suited for trigger PDEs to serve as key components in regulatory switches.This article is part of the themed issue 'The new bacteriology'.

Keywords: EAL domain; biofilm; cellulose; curli fibres; diguanylate cyclase; second messenger.

Figure 1.

Diversity of c-di-GMP-binding effectors. Proteins (circles or ovals) that belong to a variety of different protein families as well as RNAs, i.e. 5′-untranslated regions of mRNAs (riboswitches; irregularly shaped star), can bind c-di-GMP with affinities ranging over three orders of magnitude (K_d between low nanomolar and low micromolar). Classical TFs (labelled in yellow) allosterically regulated by c-di-GMP include the AAA+ ATPase FleQ [33], PelD [34] and the MerR-like regulator BrlR [35] found in Pseudomonas aeruginosa, the CRP-like virulence regulator Clp in Xanthomonas campestris [36] and BldD, a master regulator of Streptomyces development [37]. YajQ [38] and PilZ [–45] are small c-di-GMP-binding proteins or domains of larger proteins that serve as versatile adaptors or coupling factors between c-di-GMP and complex targets with diverse output functions. PgaC/D is a synthase and secretion system for the exopolysaccharide PGA in E. coli that consists of two membrane-integrated proteins whose interaction and therefore activity is stabilized by binding c-di-GMP [46]. FliI is a c-di-GMP-binding component of the P. fluorescens flagellum basal body serving as rotary export ATPase with similarly functioning homologues in other type III secretion systems [47]. While degenerate GGDEF and EAL domain proteins, which are enzymatically inactive but able to bind c-di-GMP, were recognized as c-di-GMP effectors quite a while ago [48,49], PdeR and PdeL are active PDEs now termed ‘trigger PDEs’, whose function as c-di-GMP sensing effectors has only been recently characterized [30,50] and is described in detail in §§5 and 6, respectively. Note that the array of c-di-GMP-responsive effector components is not exhaustive, but those mentioned here have been chosen to represent different families of proteins.

Figure 2.

Regulatory circuits of gene expression exerted by the trigger PDEs PdeR and PdeL of E. coli. (a) At low cellular c-di-GMP levels, PdeR inhibits DgcM and the transcription factor MlrA by direct interaction and, as a consequence, the biofilm regulator CsgD is not expressed. The fact that PdeR also inactivates the inhibitor—i.e. c-di-GMP—of its own inhibitory action on DgcM/MlrA, sets up a positive feedback loop that stabilizes the inhibition of DgcM/MlrA by PdeR (i.e. the CsgD^OFF state). When c-di-GMP levels increase (e.g. during entry into stationary phase when the RpoS-dependent DgcE is induced, whereas PdeH is no longer expressed and its cellular level decreases), binding and cleavage of c-di-GMP by PdeR releases DgcM and MlrA. This allows DgcM to act as a direct co-activator for MlrA in the transcriptional activation of the csgDEFG operon and to also produce c-di-GMP (representing a positive feedback loop that stabilizes the CsgD^ON state) [30]. As a transcription factor, CsgD directly activates the expression of the subunits of amyloid curli fibres and indirectly stimulates the production of the exopolysaccharide cellulose. CsgE, CsgF and CsgG are components of the curli secretion machinery [71]. (b) PdeL activates its own expression in a manner that is inhibited by high c-di-GMP levels. In this circuit, two nested positive feedback loops seem to accelerate a decrease of the cellular c-di-GMP levels below a certain threshold: (i) positive autoregulation of PdeL and (ii) as a PDE, PdeL inactivates its own inhibitor c-di-GMP. This circuit could allow rapid and highly efficient switching to low cellular c-di-GMP levels and therefore a rapid stop of expression or activity of biofilm-related functions [50]. For additional details, see §§5 and 6.