HD-GYP is a protein domain of unknown biochemical function implicated in bacterial signaling and regulation. In the plant pathogen Xanthomonas campestris pv. campestris , the synthesis of virulence factors and dispersal of biofilms are positively controlled by a two-component signal transduction system comprising the HD-GYP domain regulatory protein RpfG and cognate sensor RpfC and by cell–cell signaling mediated by the diffusible signal molecule DSF (diffusible signal factor). The RpfG/RpfC two-component system has been implicated in DSF perception and signal transduction. Here we show that the role of RpfG is to degrade the unusual nucleotide cyclic di-GMP, an activity associated with the HD-GYP domain. Mutation of the conserved H and D residues of the isolated HD-GYP domain resulted in loss of both the enzymatic activity against cyclic di-GMP and the regulatory activity in virulence factor synthesis. Two other protein domains, GGDEF and EAL, are already implicated in the synthesis and degradation respectively of cyclic di-GMP. As with GGDEF and EAL domains, the HD-GYP domain is widely distributed in free-living bacteria and occurs in plant and animal pathogens, as well as beneficial symbionts and organisms associated with a range of environmental niches. Identification of the role of the HD-GYP domain thus increases our understanding of a signaling network whose importance to the lifestyle of diverse bacteria is now emerging.

SummaryInterspecies signalling through the action of diffusible signal molecules can influence the behaviour of organisms growing in polymicrobial communities. Stenotrophomonas maltophilia and Pseudomonas aeruginosa occur ubiquitously in the environment and can be found together in diverse niches including the rhizosphere of plants and the cystic fibrosis lung. In mixed species biofilms, S. maltophilia substantially influenced the architecture of P. aeruginosa structures, which developed as extended filaments. This effect depended upon the synthesis of the diffusible signal factor (DSF) by S. maltophilia and could be mimicked by the addition of synthetic DSF. This response of P. aeruginosa to DSF required PA1396, a sensor kinase with an input domain of related amino acid sequence to the sensory input domain of RpfC, which is responsible for DSF perception in xanthomonads. Mutation of PA1396 or addition of DSF to P. aeruginosa led to increased levels of a number of proteins with roles in bacterial stress tolerance, including those implicated in resistance to cationic antimicrobial peptides. This effect was associated with increased tolerance to polymyxins. Homologues of PA1396 occur in a number of phytopathogenic and plant-associated pseudomonads, suggesting that modulation of bacterial behaviour through DSFmediated interspecies signalling with xanthomonads is a phenomenon that occurs widely.

Cyclic di-GMP [bis-(3Ј-5Ј)-cyclic di-GMP] (Fig. 1) is a novel second messenger in bacteria that was first described as an allosteric activator of cellulose synthase in Gluconacetobacter xylinus (49). It is now established that this nucleotide is almost ubiquitous in bacteria, where it regulates a range of functions including developmental transitions, aggregative behavior, adhesion, biofilm formation, and the virulence of animal and plant pathogens (for reviews, see references 15, 18, 30, 31, 47, and 48). The level of cyclic di-GMP in bacterial cells is influenced by both synthesis and degradation. The GGDEF protein domain synthesizes cyclic di-GMP, whereas the EAL and HD-GYP domains are involved in cyclic di-GMP hydrolysis (43,50,51,53,54,58). Bacterial genomes encode a number of proteins with GGDEF, EAL, and HD-GYP domains; e.g., in Pseudomonas aeruginosa, there are 40 such proteins. The majority of these proteins contain additional signal input domains. These signaling systems are presumed to use cyclic di-GMP as a second messenger to link the sensing of specific environmental cues to appropriate alterations in bacterial physiology and/or gene expression. Some details about the operation and organization of cyclic di-GMP signaling systems are emerging. Nevertheless, many questions remain to be answered, and new puzzles have arisen as a result of experimental investigations. Here, we review the recent advances and highlight the considerable gaps in our understanding of cyclic di-GMP signaling. In particular, we discuss (i) the protein domains involved in cyclic di-GMP turnover, highlighting HD-GYP, recently described as a novel cyclic di-GMP phosphodiesterase; (ii) signals that are transduced through cyclic di-GMP signaling; (iii) the role of cyclic di-GMP signaling in bacterial virulence; (iv) recent findings addressing the organization of, and interplay between, different cyclic di-GMP signaling systems; (v) the concept of localized pools of cyclic di-GMP within the cell; and (vi) the mechanisms by which cyclic di-GMP exerts its effects on diverse cellular functions.

The genome of Stenotrophomonas maltophilia encodes a cell-cell signaling system that is highly related to the diffusible signal factor (DSF)-dependent system of the phytopathogen Xanthomonas campestris. Here we show that in S. maltophilia, DSF signaling controls factors contributing to the virulence and antibiotic resistance of this important nosocomial pathogen.

Cyclic di-GMP is an almost ubiquitous second messenger in bacteria that was first described as an allosteric activator of cellulose synthase but is now known to regulate a range of functions, including virulence in human and animal pathogens. Two protein domains, GGDEF and EAL, are implicated in the synthesis and degradation, respectively, of cyclic di-GMP. These domains are widely distributed in bacteria, including plant pathogens. The majority of proteins with GGDEF and EAL domains contain additional signal input domains, suggesting that their activities are responsive to environmental cues. Recent studies have demonstrated that a third domain, HD-GYP, is also active in cyclic di-GMP degradation. In the plant pathogen Xanthomonas campestris pv. campestris, a two-component signal transduction system comprising the HD-GYP domain regulatory protein RpfG and cognate sensor RpfC positively controls virulence. The signals recognized by RpfC may include the cell-cell signal DSF, which also acts to regulate virulence in X. campestris pv. campestris. Here, we review these recent advances in our understanding of cyclic di-GMP signaling with particular reference to one or more roles in the bacterial pathogenesis of plants.