Signal transduction pathways often use small molecule second-messengers to integrate, amplify and transmit information to intracellular sensors and effectors (1
). Among the most important are cyclic nucleotides such as cyclic adenosine monophosphate and cyclic guanosine monophosphate which regulate a variety of functions ranging from sugar metabolism to ion channel conductance in prokaryotes and eukaryotes.
In contrast to the well-established roles of cyclic mono-nucleotides, cyclic di-nucleotides have gained prominence only recently as major prokaryotic signalling molecules. The bacterial second messenger bis-(3′–5′)-cyclic-dimeric-guanosine monophosphate (c-di-GMP), the focus of this study, was identified in 1987 as an allosteric activator of cellulose synthase in Gluconacetobacter xylinus
and Agrobacterium tumefaciens
). Since then, the molecule has become recognized as a key regulator for complex cellular functions (). Most notably, c-di-GMP controls the switch between motile and sessile lifestyles: high cellular levels of c-di-GMP promote exopolysaccharide production and surface adhesion, eventually leading to biofilm formation; conversely, low c-di-GMP levels result in flagellar gene- expression and increased cellular motility (4
). Further, there is now substantial evidence that c-di-GMP has a role beyond these functions; for example, in regulating virulence in pathogens such as Vibrio cholerae
and Pseudomonas aeruginosa
) and responding to nutrient starvation in Mycobacterium smegmatis
Figure 1. Turnover of c-di-GMP. A schematic representation of the synthesis (from GTP) and hydrolysis (to pGpG) of c-di-GMP by the GGDEF and EAL domains, respectively. Also shown are general cellular functions that are activated (arrow-shaped head) and repressed (more ...)
C-di-GMP levels are modulated through the activity of di-guanylate cyclases (DGCs) that convert two molecules of GTP to c-di-GMP, and phosphodiesterases (PDEs) that linearize c-di-GMP to pGpG, which is subsequently hydrolyzed to GMP (). DGCs are characterized by the active site GG[D/E]EF amino acid motif in the enzyme catalytic site, whereas PDEs contain either the EAL domain or the more recently described HD-GYP domain (4
). The simplest proteins involved in c-di-GMP turnover have only one of these domains: these we call GGDEF-only, EAL-only or HD-GYP-only proteins. In what has been termed a ‘biochemical conundrum’, there are also hybrid proteins containing both GGDEF and EAL domains (8
). Early biochemical studies identified such proteins in which only one of the domains was catalytically active, leading to suggestions that the other domain had acquired a regulatory function (4
). However, there are now several examples of hybrid proteins that retain both DGC and PDE activities (7
), raising questions on how the two activities are co-ordinately or reciprocally regulated.
An early computational survey of signalling proteins in 30 prokaryotic genomes showed that GGDEF and EAL proteins are ubiquitous in bacteria, but absent from archaea (8
). In general, genomes were found to encode several GGDEF and EAL proteins with a particularly striking expansion in Gamma Proteobacteria. Given that most of these proteins are likely to be mainly involved in c-di-GMP signalling, it is intriguing that organisms should encode multiple proteins containing the same enzymatic domain. This has led to questions on how the activities of different GGDEF and EAL proteins are separated in order to minimize cross-talk among distinct outputs.
The main method for managing parallel c-di-GMP signalling systems is through tight regulation of the DGC and PDE activities. The first point of regulation is at the level of transcription; however, most GGDEF and EAL proteins in Escherichia coli K12
are expressed during the stationary phase of growth, with only a few being produced during exponential growth state (12
). Thus, given that many proteins are expressed under the same condition, their spatial control and post-translational regulation are also probably critical. The genomic study above identified a number of small molecule-sensing and phosphorylation-receiving domains that frequently occur in GGDEF and EAL-containing proteins (11
) thus illustrating the importance of signal-dependent post-translational regulation. However, the prevalence of these partner domains has not been systematically investigated. Further, apart from individual examples (10
), the implications of partner domains for GGDEF-only, EAL-only and hybrid protein activity have not been discussed.
In this computational study, we investigate how cells might manage potentially detrimental cross-talk between multiple c-di-GMP signalling pathways through spatial localization and post-translational control of GGDEF- and EAL-containing proteins. In addition, we interrogate the ‘biochemical conundrum’ of hybrid proteins by investigating the prevalence of DGC and PDE activity and associated regulatory sequence motifs in such proteins. Our results complement the detailed findings from numerous molecular studies and provide a genome-scale framework for understanding c-di-GMP signalling and control.