Cyclic-di-GMP is a ubiquitous second messenger in bacteria. The recent discovery that c-di-GMP antagonistically controls motility and virulence of single, planktonic cells on one hand and cell adhesion and persistence of multicellular communities on the other has spurred interest in this regulatory compound. Cellular levels of c-di-GMP are controlled through the opposing activities of diguanylate cyclases and phosphodiesterases, which represent two large families of output domains found in bacterial one- and two-component systems. This review concentrates on structural and functional aspects of diguanylate cyclases and phosphodiesterases, and on their role in transmitting environmental stimuli into a range of different cellular functions. In addition, we examine several well-established model systems for c-di-GMP signaling, including Pseudomonas, Vibrio, Caulobacter, and Salmonella.

Background: Pseudomonas fluorescens are common soil bacteria that can improve plant health through nutrient cycling, pathogen antagonism and induction of plant defenses. The genome sequences of strains SBW25 and Pf0-1 were determined and compared to each other and with P. fluorescens Pf-5. A functional genomic in vivo expression technology (IVET) screen provided insight into genes used by P. fluorescens in its natural environment and an improved understanding of the ecological significance of diversity within this species. Results: Comparisons of three P. fluorescens genomes (SBW25, Pf0-1, Pf-5) revealed considerable divergence: 61% of genes are shared, the majority located near the replication origin. Phylogenetic and average amino acid identity analyses showed a low overall relationship. A functional screen of SBW25 defined 125 plant-induced genes including a range of functions specific to the plant environment. Orthologues of 83 of these exist in Pf0-1 and Pf-5, with 73 shared by both strains. The P. fluorescens genomes carry numerous complex repetitive DNA sequences, some resembling Miniature Inverted-repeat Transposable Elements (MITEs). In SBW25, repeat density and distribution revealed 'repeat deserts' lacking repeats, covering approximately 40% of the genome. Conclusions: P. fluorescens genomes are highly diverse. Strain-specific regions around the replication terminus suggest genome compartmentalization. The genomic heterogeneity among the three strains is reminiscent of a species complex rather than a single species. That 42% of plant-inducible genes were not shared by all strains reinforces this conclusion and shows that ecological success requires specialized and core functions. The diversity also indicates the significant size of genetic information within the Pseudomonas pan genome.

During long-term cystic fibrosis lung infections, Pseudomonas aeruginosa undergoes genetic adaptation resulting in progressively increased persistence and the generation of adaptive colony morphotypes. This includes small colony variants (SCVs), auto-aggregative, hyper-adherent cells whose appearance correlates with poor lung function and persistence of infection. The SCV morphotype is strongly linked to elevated levels of cyclic-di-GMP, a ubiquitous bacterial second messenger that regulates the transition between motile and sessile, cooperative lifestyles. A genetic screen in PA01 for SCV-related loci identified the yfiBNR operon, encoding a tripartite signaling module that regulates c-di-GMP levels in P. aeruginosa. Subsequent analysis determined that YfiN is a membrane-integral diguanylate cyclase whose activity is tightly controlled by YfiR, a small periplasmic protein, and the OmpA/Pal-like outer-membrane lipoprotein YfiB. Exopolysaccharide synthesis was identified as the principal downstream target for YfiBNR, with increased production of Pel and Psl exopolysaccharides responsible for many characteristic SCV behaviors. An yfi-dependent SCV was isolated from the sputum of a CF patient. Consequently, the effect of the SCV morphology on persistence of infection was analyzed in vitro and in vivo using the YfiN-mediated SCV as a representative strain. The SCV strain exhibited strong, exopolysaccharide-dependent resistance to nematode scavenging and macrophage phagocytosis. Furthermore, the SCV strain effectively persisted over many weeks in mouse infection models, despite exhibiting a marked fitness disadvantage in vitro. Exposure to sub-inhibitory concentrations of antibiotics significantly decreased both the number of suppressors arising, and the relative fitness disadvantage of the SCV mutant in vitro, suggesting that the SCV persistence phenotype may play a more important role during antimicrobial chemotherapy. This study establishes YfiBNR as an important player in P. aeruginosa persistence, and implicates a central role for c-di-GMP, and by extension the SCV phenotype in chronic infections. During long-term chronic infections of cystic fibrosis patients, Pseudomonas aeruginosa adapts to the lung environment, generating various different morphotypes including small colony variants (SCVs), small, strongly adherent colonies whose appearance correlates with persistence of infection. The SCV morphology is strongly associated with increased levels of the signaling molecule cyclic di-GMP. In this study we investigated the connection between cyclic di-GMP, SCV and persistence of infection. Following a genetic screen for mutants that displayed SCV morphologies, we identified and characterized the YfiBNR system. YfiN is a membrane-bound cyclic di-GMP producing enzyme, whose activity is tightly controlled by YfiR and YfiB. Cyclic di-GMP produced by YfiN boosts exopolysaccharide synthesis, generating an SCV morphotype upon YfiR-mediated release of YfiN repression. The resulting YfiN-mediated SCV morphotype is highly resistant to macrophage phagocytosis in vitro, suggesting a role for the SCV phenotype in immune system evasion. Consistent with this, YfiN de-repression increased the persistence of P. aeruginosa in long-term infections in a mouse model. The observation that the addition of antibiotics decreased the number of suppressors, and the relative fitness disadvantage of the YfiN-mediated SCV morphotype in liquid culture, suggested that SCV-mediated persistence might be favored during antimicrobial chemotherapy.

Summary: Future Challenges in Plant Pathology: Plant infection is a complicated process. On encountering a plant, pathogenic microorganisms must first adapt to life on the epiphytic surface, and survive long enough to initiate an infection. Responsiveness to the environment is critical throughout infection, with intracellular and community‐level signal transduction pathways integrating environmental signals and triggering appropriate responses in the bacterial population. Ultimately, phytopathogens must migrate from the epiphytic surface into the plant tissue using motility and chemotaxis pathways. This migration is coupled with overcoming the physical and chemical barriers to entry into the plant apoplast. Once inside the plant, bacteria use an array of secretion systems to release phytotoxins and protein effectors that fulfil diverse pathogenic functions (Fig. ) (Melotto and Kunkel, ; Phan Tran et al ., ). As our understanding of the pathways and mechanisms underpinning plant pathogenicity increases, a number of central research challenges are emerging that will profoundly shape the direction of research in the future. We need to understand the bacterial phenotypes that promote epiphytic survival and surface adaptation in pathogenic bacteria. How do these pathways function in the context of the plant‐associated microbiome, and what impact does this complex microbial community have on the onset and severity of plant infections? The huge importance of bacterial signal transduction to every stage of plant infection is becoming increasingly clear. However, there is a great deal to learn about how these signalling pathways function in phytopathogenic bacteria, and the contribution they make to various aspects of plant pathogenicity. We are increasingly able to explore the structural and functional diversity of small‐molecule natural products from plant pathogens. We need to acquire a much better understanding of the production, deployment, functional redundancy and physiological roles of these molecules. Type III secretion systems (T3SSs) are important and well‐studied contributors to bacterial disease. Several key unanswered questions will shape future investigations of these systems. We need to define the mechanism of hierarchical and temporal control of effector secretion. For successful infection, effectors need to interact with host components to exert their function. Advanced biochemical, proteomic and cell biological techniques will enable us to study the function of effectors inside the host cell in more detail and on a broader scale. Population genomics analyses provide insight into evolutionary adaptation processes of phytopathogens. The determination of the diversity and distribution of type III effectors (T3Es) and other virulence genes within and across pathogenic species, pathovars and strains will allow us to understand how pathogens adapt to specific hosts, the evolutionary pathways available to them, and the possible future directions of the evolutionary arms race between effectors and molecular plant targets. Although pathogenic bacteria employ a host of different virulence and proliferation strategies, as a result of the space constraints, this review focuses mainly on the hemibiotrophic pathogens. We discuss the process of plant infection from the perspective of these important phytopathogens, and highlight new approaches to address the outstanding challenges in this important and fast‐moving field.

The genetic adaptation of pathogens in host tissue plays a key role in the establishment of chronic infections. While whole genome sequencing has opened up the analysis of genetic changes occurring during long-term infections, the identification and characterization of adaptive traits is often obscured by a lack of knowledge of the underlying molecular processes. Our research addresses the role of Pseudomonas aeruginosa small colony variant (SCV) morphotypes in long-term infections. In the lungs of cystic fibrosis patients, the appearance of SCVs correlates with a prolonged persistence of infection and poor lung function. Formation of P. aeruginosa SCVs is linked to increased levels of the second messenger c-di-GMP. Our previous work identified the YfiBNR system as a key regulator of the SCV phenotype. The effector of this tripartite signaling module is the membrane bound diguanylate cyclase YfiN. Through a combination of genetic and biochemical analyses we first outline the mechanistic principles of YfiN regulation in detail. In particular, we identify a number of activating mutations in all three components of the Yfi regulatory system. YfiBNR is shown to function via tightly controlled competition between allosteric binding sites on the three Yfi proteins; a novel regulatory mechanism that is apparently widespread among periplasmic signaling systems in bacteria. We then show that during long-term lung infections of CF patients, activating mutations invade the population, driving SCV formation in vivo. The identification of mutational “scars” in the yfi genes of clinical isolates suggests that Yfi activity is both under positive and negative selection in vivo and that continuous adaptation of the c-di-GMP network contributes to the in vivo fitness of P. aeruginosa during chronic lung infections. These experiments uncover an important new principle of in vivo persistence, and identify the c-di-GMP network as a valid target for novel anti-infectives directed against chronic infections. Here we investigate the molecular function of the important cyclic-di-GMP signaling system YfiBNR in the opportunistic pathogen Pseudomonas aeruginosa and demonstrate its importance for the evolution of persistent small colony variant (SCV) morphotypes in chronic cystic fibrosis (CF) lung infections. Previously we showed that YfiN is a membrane bound diguanylate cyclase, whose activity is controlled by the soluble periplasmic repressor YfiR and the outer-membrane peptidoglycan binding protein YfiB. In this study we use a combination of genetic and biochemical analyses to investigate the mechanistic principles of YfiN regulation. By examining a series of activating mutations throughout the yfi operon, we show that YfiBNR functions via tightly controlled competition between allosteric binding sites on the three Yfi proteins; a novel regulatory mechanism that is apparently widespread among periplasmic signaling systems in bacteria. We then show that during long-term CF lung infections, Yfi activating mutations invade the population, driving SCV formation in vivo. The identification of mutational “scars” in the yfi genes of clinical isolates further suggests that Yfi activity is both under positive and negative selection in vivo, with Yfi-mediated SCVs acting as an environmental pool for the generation of new smooth morphotypes.