A cyclic GMP-dependent signalling pathway regulates bacterial phytopathogenesis.

Cyclic guanosine 3',5'-monophosphate (cyclic GMP) is a second messenger whose role in bacterial signalling is poorly understood. A genetic screen in the plant pathogen Xanthomonas campestris (Xcc) identified that XC_0250, which encodes a protein with a class III nucleotidyl cyclase domain, is required for cyclic GMP synthesis. Purified XC_0250 was active in cyclic GMP synthesis in vitro. The linked gene XC_0249 encodes a protein with a cyclic mononucleotide-binding (cNMP) domain and a GGDEF diguanylate cyclase domain. The activity of XC_0249 in cyclic di-GMP synthesis was enhanced by addition of cyclic GMP. The isolated cNMP domain of XC_0249 bound cyclic GMP and a structure-function analysis, directed by determination of the crystal structure of the holo-complex, demonstrated the site of cyclic GMP binding that modulates cyclic di-GMP synthesis. Mutation of either XC_0250 or XC_0249 led to a reduced virulence to plants and reduced biofilm formation in vitro. These findings describe a regulatory pathway in which cyclic GMP regulates virulence and biofilm formation through interaction with a novel effector that directly links cyclic GMP and cyclic di-GMP signalling.

The DSF Family of Cell-Cell Signals: An Expanding Class of Bacterial Virulence Regulators.

Many pathogenic bacteria use cell-cell signaling systems involving the synthesis and perception of diffusible signal molecules to control virulence as a response to cell density or confinement to niches. Bacteria produce signals of diverse structural classes. Signal molecules of the diffusible signal factor (DSF) family are cis-2-unsaturated fatty acids. The paradigm is cis-11-methyl-2-dodecenoic acid from Xanthomonas campestris pv. campestris (Xcc), which controls virulence in this plant pathogen. Although DSF synthesis was thought to be restricted to the xanthomonads, it is now known that structurally related molecules are produced by the unrelated bacteria Burkholderia cenocepacia and Pseudomonas aeruginosa. Furthermore, signaling involving these DSF family members contributes to bacterial virulence, formation of biofilms and antibiotic tolerance in these important human pathogens. Here we review the recent advances in understanding DSF signaling and its regulatory role in different bacteria. These advances include the description of the pathway/mechanism of DSF biosynthesis, identification of novel DSF synthases and new members of the DSF family, the demonstration of a diversity of DSF sensors to include proteins with a Per-Arnt-Sim (PAS) domain and the description of some of the signal transduction mechanisms that impinge on virulence factor expression. In addition, we address the role of DSF family signals in interspecies signaling that modulates the behavior of other microorganisms. Finally, we consider a number of recently reported approaches for the control of bacterial virulence through the modulation of DSF signaling.

Functional and genomic insights into the pathogenesis of Burkholderia species to rice.

A number of species of bacteria from the genus Burkholderia have been shown to be causal agents of diseases of rice. These diseases, caused by Burkholderia glumae, B. gladioli and B. plantarii, are becoming increasingly common across the globe. This is particularly so for B. glumae, whose ability to grow at elevated temperatures suggests that it may become a prevalent problem in an era of global warming. Despite the increasing threat to rice, relatively little is known about the virulence mechanisms employed by these pathogens. Work over the last 5 years has provided an increasing insight into these factors and their control by environmental and other cues. In addition, the determination of a number of genome sequences has allowed bioinformatic predictions of further possible mechanisms, which can now be investigated experimentally. Here, we review recent advances in the understanding of virulence of Burkholderia to rice, to include discussion of the roles of toxins, type II secreted enzymes, type III secreted effectors and motility as well as their regulation by quorum sensing, two-component systems and cyclic di-GMP signalling. Finally, we consider a number of approaches for the control of bacterial virulence through the modulation of quorum sensing and toxin degradation.

Novel cyclic di-GMP effectors of the YajQ protein family control bacterial virulence.

Bis-(3',5') cyclic di-guanylate (cyclic di-GMP) is a key bacterial second messenger that is implicated in the regulation of many critical processes that include motility, biofilm formation and virulence. Cyclic di-GMP influences diverse functions through interaction with a range of effectors. Our knowledge of these effectors and their different regulatory actions is far from complete, however. Here we have used an affinity pull-down assay using cyclic di-GMP-coupled magnetic beads to identify cyclic di-GMP binding proteins in the plant pathogen Xanthomonas campestris pv. campestris (Xcc). This analysis identified XC_3703, a protein of the YajQ family, as a potential cyclic di-GMP receptor. Isothermal titration calorimetry showed that the purified XC_3703 protein bound cyclic di-GMP with a high affinity (K(d)∼2 µM). Mutation of XC_3703 led to reduced virulence of Xcc to plants and alteration in biofilm formation. Yeast two-hybrid and far-western analyses showed that XC_3703 was able to interact with XC_2801, a transcription factor of the LysR family. Mutation of XC_2801 and XC_3703 had partially overlapping effects on the transcriptome of Xcc, and both affected virulence. Electromobility shift assays showed that XC_3703 positively affected the binding of XC_2801 to the promoters of target virulence genes, an effect that was reversed by cyclic di-GMP. Genetic and functional analysis of YajQ family members from the human pathogens Pseudomonas aeruginosa and Stenotrophomonas maltophilia showed that they also specifically bound cyclic di-GMP and contributed to virulence in model systems. The findings thus identify a new class of cyclic di-GMP effector that regulates bacterial virulence.

Structural Insights into the Distinct Binding Mode of Cyclic Di-AMP with SaCpaA_RCK.

Cyclic di-AMP (c-di-AMP) is a relatively new member of the family of bacterial cyclic dinucleotide second messengers. It has attracted significant attention in recent years because of the abundant roles it plays in a variety of Gram-positive bacteria. The structural features that allow diverse bacterial proteins to bind c-di-AMP are not fully understood. Here we report the biophysical and structural studies of c-di-AMP in complex with a bacterial cation-proton antiporter (CpaA) RCK (regulator of the conductance of K(+)) protein from Staphylococcus aureus (Sa). The crystal structure of the SaCpaA_RCK C-terminal domain (CTD) in complex with c-di-AMP was determined to a resolution of 1.81 Å. This structure revealed two well-liganded water molecules, each interacting with one of the adenine bases by a unique H2Olp-π interaction to stabilize the complex. Sequence blasting using the SaCpaA_RCK primary sequence against the bacterial genome database returned many CpaA analogues, and alignment of these sequences revealed that the active site residues are all well-conserved, indicating a universal c-di-AMP binding mode for CpaA_RCK. A proteoliposome activity assay using the full-length SaCpaA membrane protein indicated that c-di-AMP binding alters its antiporter activity by approximately 40%. A comparison of this structure to all other reported c-di-AMP-receptor complex structures revealed that c-di-AMP binds to receptors in either a "U-shape" or "V-shape" mode. The two adenine rings are stabilized in the inner interaction zone by a variety of CH-π, cation-π, backbone-π, or H2Olp-π interaction, but more commonly in the outer interaction zone by hydrophobic CH-π or π-π interaction. The structures determined to date provide an understanding of the mechanisms by which a single c-di-AMP can interact with a variety of receptor proteins, and how c-di-AMP binds receptor proteins in a special way different from that of c-di-GMP.

The PAS domain-containing histidine kinase RpfS is a second sensor for the diffusible signal factor of Xanthomonas campestris.

A cell-cell signalling system mediated by the fatty acid signal DSF controls the virulence of Xanthomonas campestris pv. campestris (Xcc) to plants. The synthesis and recognition of the DSF signal depends upon different Rpf proteins. DSF signal generation requires RpfF whereas signal perception and transduction depends upon the sensor RpfC and regulator RpfG. Detailed analyses of the regulatory roles of different Rpf proteins have suggested the occurrence of further sensors for DSF. Here we have used a mutagenesis approach coupled with high-resolution transcriptional analysis to identify XC_2579 (RpfS) as a second sensor for DSF in Xcc. RpfS is a complex sensor kinase predicted to have multiple Per/Arnt/Sim (PAS) domains, a histidine kinase domain and a C-terminal receiver (REC) domain. Isothermal calorimetry showed that DSF bound to the isolated N-terminal PAS domain with a Kd of 1.4 µM. RpfS controlled expression of a sub-set of genes distinct from those controlled by RpfC to include genes involved in type IV secretion and chemotaxis. Mutation of XC_2579 was associated with a reduction in virulence of Xcc to Chinese Radish when assayed by leaf spraying but not by leaf inoculation, suggesting a role for RpfS-controlled factors in the epiphytic phase of the disease cycle.

Crystal structure of an HD-GYP domain cyclic-di-GMP phosphodiesterase reveals an enzyme with a novel trinuclear catalytic iron centre.

Bis-(3',5') cyclic di-guanylate (c-di-GMP) is a key bacterial second messenger that is implicated in the regulation of many crucial processes that include biofilm formation, motility and virulence. Cellular levels of c-di-GMP are controlled through synthesis by GGDEF domain diguanylate cyclases and degradation by two classes of phosphodiesterase with EAL or HD-GYP domains. Here, we have determined the structure of an enzymatically active HD-GYP domain protein from Persephonella marina (PmGH) alone, in complex with substrate (c-di-GMP) and final reaction product (GMP). The structures reveal a novel trinuclear iron binding site, which is implicated in catalysis and identify residues involved in recognition of c-di-GMP. This structure completes the picture of all domains involved in c-di-GMP metabolism and reveals that the HD-GYP family splits into two distinct subgroups containing bi- and trinuclear metal centres.

Heterogeneity in ess transcriptional organization and variable contribution of the Ess/Type VII protein secretion system to virulence across closely related Staphylocccus aureus strains.

The Type VII protein secretion system, found in Gram-positive bacteria, secretes small proteins, containing a conserved W-x-G amino acid sequence motif, to the growth medium. Staphylococcus aureus has a conserved Type VII secretion system, termed Ess, which is dispensable for laboratory growth but required for virulence. In this study we show that there are unexpected differences in the organization of the ess gene cluster between closely related strains of S. aureus. We further show that in laboratory growth medium different strains of S. aureus secrete the EsxA and EsxC substrate proteins at different growth points, and that the Ess system in strain Newman is inactive under these conditions. Systematic deletion analysis in S. aureus RN6390 is consistent with the EsaA, EsaB, EssA, EssB, EssC and EsxA proteins comprising core components of the secretion machinery in this strain. Finally we demonstrate that the Ess secretion machinery of two S. aureus strains, RN6390 and COL, is important for nasal colonization and virulence in the murine lung pneumonia model. Surprisingly, however, the secretion system plays no role in the virulence of strain SA113 under the same conditions.

Communication, cooperation, and social interactions: a report from the third Young Microbiologists Symposium on microbe signalling, organisation, and pathogenesis.

The third Young Microbiologists Symposium took place on the vibrant campus of the University of Dundee, Scotland, from the 2nd to 3rd of June 2014. The symposium attracted over 150 microbiologists from 17 different countries. The significant characteristic of this meeting was that it was specifically aimed at providing a forum for junior scientists to present their work. The meeting was supported by the Society for General Microbiology and the American Society for Microbiology, with further sponsorship from the European Molecular Biology Organization, the Federation of European Microbiological Societies, and The Royal Society of Edinburgh. In this report, we highlight some themes that emerged from the many exciting talks and poster presentations given by the young and talented microbiologists in the area of microbial gene expression, regulation, biogenesis, pathogenicity, and host interaction.

High-resolution transcriptional analysis of the regulatory influence of cell-to-cell signalling reveals novel genes that contribute to Xanthomonas phytopathogenesis.

The bacterium Xanthomonas campestris is an economically important pathogen of many crop species and a model for the study of bacterial phytopathogenesis. In X. campestris, a regulatory system mediated by the signal molecule DSF controls virulence to plants. The synthesis and recognition of the DSF signal depends upon different Rpf proteins. DSF signal generation requires RpfF whereas signal perception and transduction depends upon a system comprising the sensor RpfC and regulator RpfG. Here we have addressed the action and role of Rpf/DSF signalling in phytopathogenesis by high-resolution transcriptional analysis coupled to functional genomics. We detected transcripts for many genes that were unidentified by previous computational analysis of the genome sequence. Novel transcribed regions included intergenic transcripts predicted as coding or non-coding as well as those that were antisense to coding sequences. In total, mutation of rpfF, rpfG and rpfC led to alteration in transcript levels (more than fourfold) of approximately 480 genes. The regulatory influence of RpfF and RpfC demonstrated considerable overlap. Contrary to expectation, the regulatory influence of RpfC and RpfG had limited overlap, indicating complexities of the Rpf signalling system. Importantly, functional analysis revealed over 160 new virulence factors within the group of Rpf-regulated genes.

Proteomics analysis of the regulatory role of Rpf/DSF cell-to-cell signaling system in the virulence of Xanthomonas campestris.

The black rot pathogen Xanthomonas campestris utilizes molecules of the diffusible signal factor (DSF) family as signals to regulate diverse processes contributing to virulence. DSF signal synthesis and transduction requires proteins encoded by the rpf gene cluster. RpfF catalyzes DSF synthesis, whereas the RpfCG two-component system links the perception of DSF to alteration in the level of the second messenger cyclic di-GMP. As this nucleotide can exert a regulatory influence at the post-transcriptional and post-translational levels, we have used comparative proteomics to identify Rpf-regulated processes in X. campestris that may not be revealed by transcriptomics. The abundance of a number of proteins was altered in rpfF, rpfC, or rpfG mutants compared with the wild type. These proteins belonged to several functional categories, including biosynthesis and intermediary metabolism, regulation, oxidative stress or antibiotic resistance, and DNA replication. For many of these proteins, the alteration in abundance was not associated with alteration in transcript level. A directed mutational analysis allowed us to describe a number of new virulence factors among these proteins, including elongation factor P and a putative outer membrane protein, which are both widely conserved in bacteria.

Real-time electrochemical detection of ATP and H2O2 release in freshly isolated kidneys.

Extracellular nucleotides such as adenosine-5'-triphosphate (ATP) and reactive oxygen species are essential local signaling molecules in the kidney. However, measurements of changes in the interstitial concentrations of these substances in response to various stimuli remain hindered due to limitations of existing experimental techniques. The goal of this study was to develop a novel approach suitable for real-time measurements of ATP and H2O2 levels in freshly isolated rat kidney. Rats were anesthetized and the kidneys were flushed to clear blood before isolation for consequent perfusion. The perfused kidneys were placed into a bath solution and dual simultaneous amperometric recordings were made with the enzymatic microelectrode biosensors detecting ATP and H2O2. It was found that basal levels of H2O2 were increased in Dahl salt-sensitive (SS) rats fed a high-salt diet compared with SS and Sprague-Dawley rats fed a low-salt diet and that medulla contained higher levels of H2O2 compared with cortex in both strains. In contrast, ATP levels did not change in SS rats when animals were fed a high-salt diet. Importantly, angiotensin II via AT1 receptor induced rapid release of both ATP and H2O2 and this effect was enhanced in SS rats. These results demonstrate that ATP and H2O2 are critical in the development of salt-sensitive hypertension and that the current method represents a unique powerful approach for the real-time monitoring of the changes in endogenous substance levels in whole organs.

Socializing, networking and development: a report from the second 'Young Microbiologists Symposium on Microbe Signalling, Organization and Pathogenesis'.

In mid-June, the second Young Microbiologists Symposium took place under the broad title of 'Microbe signalling, organization and pathogenesis' on the picturesque campus of University College Cork, Ireland. The symposium attracted 150 microbiologists from 15 different countries. The key feature of this meeting was that it was specifically aimed at providing a platform for junior scientists to present their work to a broad audience. The meeting was principally supported by Science Foundation Ireland with further backing from the Society for General Microbiology, the American Society for Microbiology and the European Molecular Biology Organization. Sessions focused on microbial gene expression, biogenesis, pathogenicity and host interaction. In this MicroMeeting report, we highlight some of the most significant advances and exciting developments reported during various talks and poster presentations given by the young and talented microbiologists.

Dynamic complex formation between HD-GYP, GGDEF and PilZ domain proteins regulates motility in Xanthomonas campestris.

RpfG is a member of a class of wide spread bacterial two-component regulators with an HD-GYP cyclic di-GMP phosphodiesterase domain. In the plant pathogen Xanthomonas campestris, RpfG together with the sensor kinase RpfC regulates multiple factors as a response to the cell-to-cell Diffusible Signalling Factor (DSF). A dynamic physical interaction of RpfG with two diguanylate cyclase (GGDEF) domain proteins controls motility. Here we show that, contrary to expectation, regulation of motility by the GGDEF domain proteins does not depend upon their cyclic di-GMP synthetic activity. Furthermore we show that the complex of RpfG and GGDEF domain proteins recruits a specific PilZ domain 'adaptor' protein, and this complex then interacts with the pilus motor proteins PilU and PiIT. The results support a model in which DSF signalling influences motility through the highly regulated dynamic interaction of proteins that affect pilus action. A specific motif that we identify to be required for HD-GYP domain interaction is conserved in a number of GGDEF domain proteins, suggesting that regulation via interdomain interactions is of broad relevance.

Cyclic di-GMP signalling and the regulation of bacterial virulence.

Signal transduction pathways involving the second messenger cyclic di-GMP [bis-(3'-5')-cyclic di-guanosine monophosphate] occur widely in bacteria where they act to link perception of environmental or intracellular cues and signals to specific alterations in cellular function. Such alterations can contribute to bacterial lifestyle transitions including biofilm formation and virulence. The cellular levels of the nucleotide are controlled through the opposing activities of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs). The GGDEF domain of DGCs catalyses the synthesis of cyclic di-GMP from GTP, whereas EAL or HD-GYP domains in different classes of PDE catalyse cyclic di-GMP degradation to pGpG and GMP. We are now beginning to understand how alterations in cyclic di-GMP exert a regulatory action through binding to diverse receptors or effectors that include a small 'adaptor' protein domain called PilZ, transcription factors and riboswitches. The regulatory action of enzymically active cyclic di-GMP signalling proteins is, however, not restricted to an influence on the level of nucleotide. Here, I will discuss our recent findings that highlight the role that protein-protein interactions involving these signalling proteins have in regulating functions that contribute to bacterial virulence.

Comparative transcriptomic analysis of the Burkholderia cepacia tyrosine kinase bceF mutant reveals a role in tolerance to stress, biofilm formation, and virulence.

The bacterial tyrosine-kinase (BY-kinase) family comprises the major group of bacterial enzymes endowed with tyrosine kinase activity. We previously showed that the BceF protein from Burkholderia cepacia IST408 belongs to this BY-kinase family and is involved in the biosynthesis of the exopolysaccharide cepacian. However, little is known about the extent of regulation of this protein kinase activity. In order to examine this regulation, we performed a comparative transcriptome profile between the bceF mutant and wild-type B. cepacia IST408. The analyses led to identification of 630 genes whose expression was significantly changed. Genes with decreased expression in the bceF mutant were related to stress response, motility, cell adhesion, and carbon and energy metabolism. Genes with increased expression were related to intracellular signaling and lipid metabolism. Mutation of bceF led to reduced survival under heat shock and UV light exposure, reduced swimming motility, and alteration in biofilm architecture when grown in vitro. Consistent with some of these phenotypes, the bceF mutant demonstrated elevated levels of cyclic-di-GMP. Furthermore, BceF contributed to the virulence of B. cepacia for larvae of the Greater wax moth, Galleria mellonella. Taken together, BceF appears to play a considerable role in many cellular processes, including biofilm formation and virulence. As homologues of BceF occur in a number of pathogenic and plant-associated Burkholderia strains, the modulation of bacterial behavior through tyrosine kinase activity is most likely a widely occurring phenomenon.

Cell-cell signal-dependent dynamic interactions between HD-GYP and GGDEF domain proteins mediate virulence in Xanthomonas campestris.

RpfG is a paradigm for a class of widespread bacterial two-component regulators with a CheY-like receiver domain attached to a histidine-aspartic acid-glycine-tyrosine-proline (HD-GYP) cyclic di-GMP phosphodiesterase domain. In the plant pathogen Xanthomonas campestris pv. campestris (Xcc), a two-component system comprising RpfG and the complex sensor kinase RpfC is implicated in sensing and responding to the diffusible signaling factor (DSF), which is essential for cell-cell signaling. RpfF is involved in synthesizing DSF, and mutations of rpfF, rpfG, or rpfC lead to a coordinate reduction in the synthesis of virulence factors such as extracellular enzymes, biofilm structure, and motility. Using yeast two-hybrid analysis and fluorescence resonance energy transfer experiments in Xcc, we show that the physical interaction of RpfG with two proteins with diguanylate cyclase (GGDEF) domains controls a subset of RpfG-regulated virulence functions. RpfG interactions were abolished by alanine substitutions of the three residues of the conserved GYP motif in the HD-GYP domain. Changing the GYP motif or deletion of the two GGDEF-domain proteins reduced Xcc motility but not the synthesis of extracellular enzymes or biofilm formation. RpfG-GGDEF interactions are dynamic and depend on DSF signaling, being reduced in the rpfF mutant but restored by DSF addition. The results are consistent with a model in which DSF signal transduction controlling motility depends on a highly regulated, dynamic interaction of proteins that influence the localized expression of cyclic di-GMP.