The gap gene of Rhizobium etli is required for both free life and symbiosis with common beans.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH or Gap) is a ubiquitous enzyme essential for carbon and energy metabolism in most organisms. Despite its primary role in sugar metabolism, GAPDH is recognized for its involvement in diverse cellular processes, being considered a paradigm among multifunctional/moonlighting proteins. Besides its canonical cytoplasmic location, GAPDH has been detected on cell surfaces or as a secreted protein in prokaryotes, yet little is known about its possible roles in plant symbiotic bacteria. Here we report that Rhizobium etli, a nitrogen-fixing symbiont of common beans, carries a single gap gene responsible for both GAPDH glycolytic and gluconeogenic activities. An active Gap protein is required throughout all stages of the symbiosis between R. etli and its host plant Phaseolus vulgaris. Both glycolytic and gluconeogenic Gap metabolic activities likely contribute to bacterial fitness during early and intermediate stages of the interaction, whereas GAPDH gluconeogenic activity seems critical for nodule invasion and nitrogen fixation. Although the R. etli Gap protein is secreted in a c-di-GMP related manner, no involvement of the R. etli gap gene in c-di-GMP related phenotypes, such as flocculation, biofilm formation or EPS production, was observed. Notably, the R. etli gap gene fully complemented a double gap1/gap2 mutant of Pseudomonas syringae for free life growth, albeit only partially in planta, suggesting potential specific roles for each type of Gap protein. Nevertheless, further research is required to unravel additional functions of the R. etli Gap protein beyond its essential metabolic roles.

FleQ, FleN and c-di-GMP coordinately regulate cellulose production in Pseudomonas syringae pv. tomato DC3000.

The second messenger cyclic di-GMP (c-di-GMP) controls the transition between motility and sessility in many bacterial species by a variety of mechanisms, including the production of multiple exopolysaccharides. Pseudomonas syringae pv. tomato (Pto) DC3000 is a plant pathogenic bacteria able to synthesize acetylated cellulose under high c-di-GMP levels thanks to the expression of the wssABCDEFGHI operon. Increased cellulose production enhances air-liquid biofilm formation and generates a wrinkled colony phenotype on solid media. We previously showed that under low levels of c-di-GMP, the regulators FleQ and AmrZ bound to adjacent sequences at the wss promoter inhibiting its expression, but only FleQ responded to the presence of c-di-GMP by activating cellulose production. In the present work, we advance in the knowledge of this complex regulation in Pto DC3000 by shedding light over the role of FleN in this process. The distinctive features of this system are that FleN and FleQ are both required for repression and activation of the wss operon under low and high c-di-GMP levels, respectively. We have also identified three putative FleQ binding sites at the wss promoter and show that FleQ/FleN-ATP binds at those sites under low c-di-GMP levels, inducing a distortion of DNA, impairing RNA polymerase binding, and repressing wss transcription. However, binding of c-di-GMP induces a conformational change in the FleQ/FleN-ATP complex, which relieves the DNA distortion, allows promoter access to the RNA polymerase, and leads to activation of wss transcription. On the other hand, AmrZ is always bound at the wss promoter limiting its expression independently of FleQ, FleN and c-di-GMP levels.

Distinctive features of the Gac-Rsm pathway in plant-associated Pseudomonas.

Productive plant-bacteria interactions, either beneficial or pathogenic, require that bacteria successfully sense, integrate and respond to continuously changing environmental and plant stimuli. They use complex signal transduction systems that control a vast array of genes and functions. The Gac-Rsm global regulatory pathway plays a key role in controlling fundamental aspects of the apparently different lifestyles of plant beneficial and phytopathogenic Pseudomonas as it coordinates adaptation and survival while either promoting plant health (biocontrol strains) or causing disease (pathogenic strains). Plant-interacting Pseudomonas stand out for possessing multiple Rsm proteins and Rsm RNAs, but the physiological significance of this redundancy is not yet clear. Strikingly, the components of the Gac-Rsm pathway and the controlled genes/pathways are similar, but the outcome of its regulation may be opposite. Therefore, identifying the target mRNAs bound by the Rsm proteins and their mode of action (repression or activation) is essential to explain the resulting phenotype. Some technical considerations to approach the study of this system are also given. Overall, several important features of the Gac-Rsm cascade are now understood in molecular detail, particularly in Pseudomonas protegens CHA0, but further questions remain to be solved in other plant-interacting Pseudomonas.

Exploring the expression and functionality of the rsm sRNAs in Pseudomonas syringae pv. tomato DC3000.

The Gac-rsm pathway is a global regulatory network that governs mayor lifestyle and metabolic changes in gamma-proteobacteria. In a previous study, we uncovered the role of CsrA proteins promoting growth and repressing motility, alginate production and virulence in the model phytopathogen Pseudomonas syringae pv. tomato (Pto) DC3000. Here, we focus on the expression and regulation of the rsm regulatory sRNAs, since Pto DC3000 exceptionally has seven variants (rsmX1-5, rsmY and rsmZ). The presented results offer further insights into the functioning of the complex Gac-rsm pathway and the interplay among its components. Overall, rsm expressions reach maximum levels at high cell densities, are unaffected by surface detection, and require GacA for full expression. The rsm levels of expression and GacA-dependence are determined by the sequences found in their -35/-10 promoter regions and GacA binding boxes, respectively. rsmX5 stands out for being the only rsm in Pto DC3000 whose high expression does not require GacA, constituting the main component of the total rsm pool in a gacA mutant. The deletion of rsmY and rsmZ had minor effects on Pto DC3000 motility and virulence phenotypes, indicating that rsmX1-5 can functionally replace them. On the other hand, rsmY or rsmZ overexpression in a gacA mutant did not revert its phenotype. Additionally, a negative feedback regulatory loop in which the CsrA3 protein promotes its own titration by increasing the levels of several rsm RNAs in a GacA-dependent manner has been disclosed as part of this work.

AmrZ and FleQ Co-regulate Cellulose Production in Pseudomonas syringae pv. Tomato DC3000.

Pseudomonas syringae pv. tomato DC3000 carries the wssABCDEFGHI operon for the synthesis of acetylated cellulose, whose production is stimulated by increasing the intracellular levels of the second messenger c-di-GMP. This enhances air-liquid biofilm formation and generates a wrinkly colony morphotype in solid media. In the present study we show that cellulose production is a complex process regulated at multiple levels and involving different players in this bacterium. Using different in vitro approaches, including Electrophoretic Mobility Shift Assay (EMSA) and footprint analysis, we demonstrated the interrelated role of two transcriptional regulators, AmrZ and FleQ, over cellulose production in Pto DC3000 and the influence of c-di-GMP in this process. Under physiological c-di-GMP levels, both regulators bind directly to adjacent regions at the wss promoter inhibiting its expression. However, just FleQ responds to c-di-GMP releasing from its wss operator site and converting from a repressor to an activator of cellulose production. The additive effect of the double amrZ/fleQ mutation on the expression of wss, together with the fact that they are not cross-regulated at the transcriptional level, suggest that FleQ and AmrZ behave as independent regulators, unlike what has been described in other Pseudomonas species. Furthermore, this dual co-regulation exerted by AmrZ and FleQ is not limited to cellulose production, but also affects other important phenotypes in Pto DC3000, such as motility and virulence.

Visualization and characterization of Pseudomonas syringae pv. tomato DC3000 pellicles.

Cellulose, whose production is controlled by c-di-GMP, is a commonly found exopolysaccharide in bacterial biofilms. Pseudomonas syringae pv. tomato (Pto) DC3000, a model organism for molecular studies of plant-pathogen interactions, carries the wssABCDEFGHI operon for the synthesis of acetylated cellulose. The high intracellular levels of the second messenger c-di-GMP induced by the overexpression of the heterologous diguanylate cyclase PleD stimulate cellulose production and enhance air-liquid biofilm (pellicle) formation. To characterize the mechanisms involved in Pto DC3000 pellicle formation, we studied this process using mutants lacking flagella, biosurfactant or different extracellular matrix components, and compared the pellicles produced in the absence and in the presence of PleD. We have discovered that neither alginate nor the biosurfactant syringafactin are needed for their formation, whereas cellulose and flagella are important but not essential. We have also observed that the high c-di-GMP levels conferred more cohesion to Pto cells within the pellicle and induced the formation of intracellular inclusion bodies and extracellular fibres and vesicles. Since the pellicles were very labile and this greatly hindered their handling and processing for microscopy, we have also developed new methods to collect and process them for scanning and transmission electron microscopy. These techniques open up new perspectives for the analysis of fragile biofilms in other bacterial strains.

Suppression of UV-B stress induced flavonoids by biotic stress: Is there reciprocal crosstalk?

Plants respond to abiotic UV-B stress with enhanced expression of genes for flavonoid production, especially the key-enzyme chalcone synthase (CHS). Some flavonoids are antioxidative, antimicrobial and/or UV-B protective secondary metabolites. However, when plants are challenged with concomitant biotic stress (simulated e.g. by the bacterial peptide flg22, which induces MAMP triggered immunity, MTI), the production of flavonoids is strongly suppressed in both Arabidopsis thaliana cell cultures and plants. On the other hand, flg22 induces the production of defense related compounds, such as the phytoalexin scopoletin, as well as lignin, a structural barrier thought to restrict pathogen spread within the host tissue. Since all these metabolites require the precursor phenylalanine for their production, suppression of the flavonoid production appears to allow the plant to focus its secondary metabolism on the production of pathogen defense related compounds during MTI. Interestingly, several flavonoids have been reported to display anti-microbial activities. For example, the plant flavonoid phloretin targets the Pseudomonas syringae virulence factors flagella and type 3 secretion system. That is, suppression of flavonoid synthesis during MTI might have also negative side-effects on the pathogen defense. To clarify this issue, we deployed an Arabidopsis flavonoid mutant and obtained genetic evidence that flavonoids indeed contribute to ward off the virulent bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. Finally, we show that UV-B attenuates expression of the flg22 receptor FLS2, indicating that there is negative and reciprocal interaction between this abiotic stress and the plant-pathogen defense responses.

Multiple CsrA Proteins Control Key Virulence Traits in Pseudomonas syringae pv. tomato DC3000.

The phytopathogenic bacterium Pseudomonas syringae pv. tomato DC3000 has a complex Gac-rsm global regulatory pathway that controls virulence, motility, production of secondary metabolites, carbon metabolism, and quorum sensing. However, despite the fact that components of this pathway are known, their physiological roles have not yet been established. Regarding the CsrA/RsmA type proteins, five paralogs, three of which are well conserved within the Pseudomonas genus (csrA1, csrA2, and csrA3), have been found in the DC3000 genome. To decipher their function, mutants lacking the three most conserved CsrA proteins have been constructed and their physiological outcomes examined. We show that they exert nonredundant functions and demonstrate that CsrA3 and, to a lesser extent, CsrA2 but not CsrA1 alter the expression of genes involved in a variety of pathways and systems important for motility, exopolysaccharide synthesis, growth, and virulence. Particularly, alginate synthesis, syringafactin production, and virulence are considerably de-repressed in a csrA3 mutant, whereas growth in planta is impaired. We propose that the linkage of growth and symptom development is under the control of CsrA3, which functions as a pivotal regulator of the DC3000 life cycle, repressing virulence traits and promoting cell division in response to environmental cues.

A novel c-di-GMP binding domain in glycosyltransferase BgsA is responsible for the synthesis of a mixed-linkage ß-glucan.

BgsA is the glycosyltransferase (GT) involved in the synthesis of a linear mixed-linkage ß-glucan (MLG), a recently described exopolysaccharide activated by c-di-GMP in Sinorhizobium meliloti and other Rhizobiales. Although BgsA displays sequence and structural homology with bacterial cellulose synthases (CS), it does not contain any predictable c-di-GMP binding domain. In this work we demonstrate that the cytoplasmic C-terminal domain of BgsA (C-BgsA) binds c-di-GMP with both high affinity (KD = 0.23 µM) and specificity. C-BgsA is structurally different to the otherwise equivalent cytoplasmic C-terminal domain of CS, and does not contain PilZ motifs for c-di-GMP recognition. A combination of random and site-directed mutagenesis with surface plasmon resonance (SPR) allowed identification of the C-BgsA residues which are important not only for c-di-GMP binding, but also for BgsA GT activity. The results suggest that the C-BgsA domain is important for both, c-di-GMP binding and GT activity of BgsA. In contrast to bacterial CS where c-di-GMP has been proposed as a derepressor of GT activity, we hypothesize that the C-terminal domain of BgsA plays an active role in BgsA GT activity upon binding c-di-GMP.

AmrZ regulates cellulose production in Pseudomonas syringae pv. tomato DC3000.

In Pseudomonas syringae pv. tomato DC3000, the second messenger c-di-GMP has been previously shown to stimulate pellicle formation and cellulose biosynthesis. A screen for genes involved in cellulose production under high c-di-GMP intracellular levels led to the identification of insertions in two genes, wssB and wssE, belonging to the Pto DC3000 cellulose biosynthesis operon wssABCDEFGHI. Interestingly, beside cellulose-deficient mutants, colonies with a rougher appearance than the wild type also arouse among the transposants. Those mutants carry insertions in amrZ, a gene encoding a transcriptional regulator in different Pseudomonas. Here, we provide evidence that AmrZ is involved in the regulation of bacterial cellulose production at transcriptional level by binding to the promoter region of the wssABCDEFGHI operon and repressing cellulose biosynthesis genes. Mutation of amrZ promotes wrinkly colony morphology, increased cellulose production and loss of motility in Pto DC3000. AmrZ regulon includes putative c-di-GMP metabolising proteins, like AdcA and MorA, which may also impact those phenotypes. Furthermore, an amrZ but not a cellulose-deficient mutant turned out to be impaired in pathogenesis, indicating that AmrZ is a key regulator of Pto DC3000 virulence probably by controlling bacterial processes other than cellulose production.

Mini-Tn7 vectors for stable expression of diguanylate cyclase PleD* in Gram-negative bacteria.

BACKGROUND: The cyclic diguanylate (c-di-GMP) is currently considered an ubiquitous second messenger in bacteria that influences a wide range of cellular processes. One of the methodological approaches to unravel c-di-GMP regulatory networks involves raising the c-di-GMP intracellular levels, e.g. by expressing a diguanylate cyclase (DGC), to provoke phenotypic changes. RESULTS: We have constructed mini-Tn7 delivery vectors for the integration and stable expression of the pleD* gene encoding a highly active DGC, which can be used to artificially increase the intracellular levels of c-di-GMP in Gram negative bacteria. The functionality of these new vectors has been validated in several plant-interacting α- and γ-proteobacteria. Similarly to vector plasmid-borne pleD*, the genome-borne mini-Tn7pleD* constructs provide significant increases in intracellular c-di-GMP, provoking expected phenotypic changes such as enhanced polysaccharide production, biofilm formation and reduced motility. However, the mini-Tn7pleD* constructs resulted far more stable in the absence of antibiotics than the plasmid-based pleD* constructs. Furthermore, we have also implemented an inducible system to modulate pleD* expression and intracellular c-di-GMP rises "on demand". CONCLUSIONS: mini-Tn7pleD* constructs are very stable and are maintained during bacterial free-living growth as well as during interaction with eukaryotic hosts, in the absence of selective pressure. This high stability ensures experimental homogeneity in time and space with regard to enhancing c-di-GMP intracellular levels in bacteria of interest.

FleQ coordinates flagellum-dependent and -independent motilities in Pseudomonas syringae pv. tomato DC3000.

Motility plays an essential role in bacterial fitness and colonization in the plant environment, since it favors nutrient acquisition and avoidance of toxic substances, successful competition with other microorganisms, the ability to locate the preferred hosts, access to optimal sites within them, and dispersal in the environment during the course of transmission. In this work, we have observed that the mutation of the flagellar master regulatory gene, fleQ, alters bacterial surface motility and biosurfactant production, uncovering a new type of motility for Pseudomonas syringae pv. tomato DC3000 on semisolid surfaces. We present evidence that P. syringae pv. tomato DC3000 moves over semisolid surfaces by using at least two different types of motility, namely, swarming, which depends on the presence of flagella and syringafactin, a biosurfactant produced by this strain, and a flagellum-independent surface spreading or sliding, which also requires syringafactin. We also show that FleQ activates flagellum synthesis and negatively regulates syringafactin production in P. syringae pv. tomato DC3000. Finally, it was surprising to observe that mutants lacking flagella or syringafactin were as virulent as the wild type, and only the simultaneous loss of both flagella and syringafactin impairs the ability of P. syringae pv. tomato DC3000 to colonize tomato host plants and cause disease.

Contribution of the non-effector members of the HrpL regulon, iaaL and matE, to the virulence of Pseudomonas syringae pv. tomato DC3000 in tomato plants.

BACKGROUND: The phytohormone indole-3-acetic acid (IAA) is widely distributed among plant-associated bacteria. Certain strains of the Pseudomonas syringae complex can further metabolize IAA into a less biologically active amino acid conjugate, 3-indole-acetyl-ε-L-lysine, through the action of the iaaL gene. In P. syringae and Pseudomonas savastanoi strains, the iaaL gene is found in synteny with an upstream gene, here called matE, encoding a putative MATE family transporter. In P. syringae pv. tomato (Pto) DC3000, a pathogen of tomato and Arabidopsis plants, the HrpL sigma factor controls the expression of a suite of virulence-associated genes via binding to hrp box promoters, including that of the iaaL gene. However, the significance of HrpL activation of the iaaL gene in the virulence of Pto DC3000 is still unclear. RESULTS: A conserved hrp box motif is found upstream of the iaaL gene in the genomes of P. syringae strains. However, although the promoter region of matE is only conserved in genomospecies 3 of this bacterial group, we showed that this gene also belongs to the Pto DC3000 HrpL regulon. We also demonstrated that the iaaL gene is transcribed both independently and as part of an operon with matE in this pathogen. Deletion of either the iaaL or the matE gene resulted in reduced fitness and virulence of Pto DC3000 in tomato plants. In addition, we used multicolor fluorescence imaging to visualize the responses of tomato plants to wild-type Pto DC3000 and to its ΔmatE and ΔiaaL mutants. Activation of secondary metabolism prior to the development of visual symptoms was observed in tomato leaves after bacterial challenges with all strains. However, the observed changes were strongest in plants challenged by the wild-type strain, indicating lower activation of secondary metabolism in plants infected with the ΔmatE or ΔiaaL mutants. CONCLUSIONS: Our results provide new evidence for the roles of non-type III effector genes belonging to the Pto DC3000 HrpL regulon in virulence.

Diguanylate cyclase DgcP is involved in plant and human Pseudomonas spp. infections.

The second messenger cyclic di-GMP (c-di-GMP) controls the transition between different lifestyles in bacterial pathogens. Here, we report the identification of DgcP (diguanylate cyclase conserved in Pseudomonads), whose activity in the olive tree pathogen Pseudomonas savastanoi pv. savastanoi is dependent on the integrity of its GGDEF domain. Furthermore, deletion of the dgcP gene revealed that DgcP negatively regulates motility and positively controls biofilm formation in both the olive tree pathogen P. savastanoi pv. savastanoi and the human opportunistic pathogen Pseudomonas aeruginosa. Overexpression of the dgcP gene in P. aeruginosa PAK led to increased exopolysaccharide production and upregulation of the type VI secretion system; in turn, it repressed the type III secretion system, which is a hallmark of chronic infections and persistence for P. aeruginosa. Deletion of the dgcP gene in P. savastanoi pv. savastanoi NCPPB 3335 and P. aeruginosa PAK reduced their virulence in olive plants and in a mouse acute lung injury model respectively. Our results show that diguanylate cyclase DgcP is a conserved Pseudomonas protein with a role in virulence, and confirm the existence of common c-di-GMP signalling pathways that are capable of regulating plant and human Pseudomonas spp. infections.

Novel mixed-linkage ß-glucan activated by c-di-GMP in Sinorhizobium meliloti.

An artificial increase of cyclic diguanylate (c-di-GMP) levels in Sinorhizobium meliloti 8530, a bacterium that does not carry known cellulose synthesis genes, leads to overproduction of a substance that binds the dyes Congo red and calcofluor. Sugar composition and methylation analyses and NMR studies identified this compound as a linear mixed-linkage (1 → 3)(1 → 4)-ß-D-glucan (ML ß-glucan), not previously described in bacteria but resembling ML ß-glucans found in plants and lichens. This unique polymer is hydrolyzed by the specific endoglucanase lichenase, but, unlike lichenan and barley glucan, it generates a disaccharidic → 4)-ß-D-Glcp-(1 → 3)-ß-D-Glcp-(1 → repeating unit. A two-gene operon bgsBA required for production of this ML ß-glucan is conserved among several genera within the order Rhizobiales, where bgsA encodes a glycosyl transferase with domain resemblance and phylogenetic relationship to curdlan synthases and to bacterial cellulose synthases. ML ß-glucan synthesis is subjected to both transcriptional and posttranslational regulation. bgsBA transcription is dependent on the exopolysaccharide/quorum sensing ExpR/SinI regulatory system, and posttranslational regulation seems to involve allosteric activation of the ML ß-glucan synthase BgsA by c-di-GMP binding to its C-terminal domain. To our knowledge, this is the first report on a linear mixed-linkage (1 → 3)(1 → 4)-ß-glucan produced by a bacterium. The S. meliloti ML ß-glucan participates in bacterial aggregation and biofilm formation and is required for efficient attachment to the roots of a host plant, resembling the biological role of cellulose in other bacteria.

The c-di-GMP phosphodiesterase BifA is involved in the virulence of bacteria from the Pseudomonas syringae complex.

In a recent screen for novel virulence factors involved in the interaction between Pseudomonas savastanoi pv. savastanoi and the olive tree, a mutant was selected that contained a transposon insertion in a putative cyclic diguanylate (c-di-GMP) phosphodiesterase-encoding gene. This gene displayed high similarity to bifA of Pseudomonas aeruginosa and Pseudomonas putida. Here, we examined the role of BifA in free-living and virulence-related phenotypes of two bacterial plant pathogens in the Pseudomonas syringae complex, the tumour-inducing pathogen of woody hosts, P. savastanoi pv. savastanoi NCPPB 3335, and the pathogen of tomato and Arabidopsis, P. syringae pv. tomato DC3000. We showed that deletion of the bifA gene resulted in decreased swimming motility of both bacteria and inhibited swarming motility of DC3000. In contrast, overexpression of BifA in P. savastanoi pv. savastanoi had a positive impact on swimming motility and negatively affected biofilm formation. Deletion of bifA in NCPPB 3335 and DC3000 resulted in reduced fitness and virulence of the microbes in olive (NCPPB 3335) and tomato (DC3000) plants. In addition, real-time monitoring of olive plants infected with green fluorescent protein (GFP)-tagged P. savastanoi cells displayed an altered spatial distribution of mutant ΔbifA cells inside olive knots compared with the wild-type strain. All free-living phenotypes that were altered in both ΔbifA mutants, as well as the virulence of the NCPPB 3335 ΔbifA mutant in olive plants, were fully rescued by complementation with P. aeruginosa BifA, whose phosphodiesterase activity has been demonstrated. Thus, these results suggest that P. syringae and P. savastanoi BifA are also active phosphodiesterases. This first demonstration of the involvement of a putative phosphodiesterase in the virulence of the P. syringae complex provides confirmation of the role of c-di-GMP signalling in the virulence of this group of plant pathogens.

Responses to elevated c-di-GMP levels in mutualistic and pathogenic plant-interacting bacteria.

Despite a recent burst of research, knowledge on c-di-GMP signaling pathways remains largely fragmentary and molecular mechanisms of regulation and even c-di-GMP targets are yet unknown for most bacteria. Besides genomics or bioinformatics, accompanying alternative approaches are necessary to reveal c-di-GMP regulation in bacteria with complex lifestyles. We have approached this study by artificially altering the c-di-GMP economy of diverse pathogenic and mutualistic plant-interacting bacteria and examining the effects on the interaction with their respective host plants. Phytopathogenic Pseudomonas and symbiotic Rhizobium strains with enhanced levels of intracellular c-di-GMP displayed common free-living responses: reduction of motility, increased production of extracellular polysaccharides and enhanced biofilm formation. Regarding the interaction with the host plants, P. savastanoi pv. savastanoi cells containing high c-di-GMP levels formed larger knots on olive plants which, however, displayed reduced necrosis. In contrast, development of disease symptoms in P. syringae-tomato or P. syringae-bean interactions did not seem significantly affected by high c-di-GMP. On the other hand, increasing c-di-GMP levels in symbiotic R. etli and R. leguminosarum strains favoured the early stages of the interaction since enhanced adhesion to plant roots, but decreased symbiotic efficiency as plant growth and nitrogen contents were reduced. Our results remark the importance of c-di-GMP economy for plant-interacting bacteria and show the usefulness of our approach to reveal particular stages during plant-bacteria associations which are sensitive to changes in c-di-GMP levels.

Plant flavonoids target Pseudomonas syringae pv. tomato DC3000 flagella and type III secretion system.

Flavonoids are among the most abundant plant secondary metabolites involved in plant protection against pathogens, but micro-organisms have developed resistance mechanisms to those compounds. We previously demonstrated that the MexAB-OprM efflux pump mediates resistance of Pseudomonas syringae pv. tomato (Pto) DC3000 to flavonoids, facilitating its survival and the colonization of the host. Here, we have shown that tomato plants respond to Pto infection producing flavonoids and other phenolic compounds. The effects of flavonoids on key traits of this model plant-pathogen bacterium have also been investigated observing that they reduce Pto swimming and swarming because of the loss of flagella, and also inhibited the expression and assembly of a functional type III secretion system. Those effects were more severe in a mutant lacking the MexAB-OprM pump. Our results suggest that flavonoids inhibit the function of the GacS/GacA two-component system, causing a depletion of rsmY RNA, therefore affecting the synthesis of two important virulence factors in Pto DC3000, flagella and the type III secretion system. These data provide new insights into the flavonoid role in the molecular dialog between host and pathogen.

Induction of Pseudomonas syringae pv. tomato DC3000 MexAB-OprM multidrug efflux pump by flavonoids is mediated by the repressor PmeR.

In this study, we have analyzed the expression of the Pseudomonas syringae pv. tomato DC3000 mexAB-oprM efflux pump operon and of the regulatory gene pmeR, and we have investigated the role of the PmeR protein on transcription from both promoters. We demonstrate that mexAB-oprM and pmeR are expressed in vivo at a relatively high and moderate basal level, respectively, which, in both cases, increases in the presence of different flavonoids and other compounds, such as butyl and methylparaben. We show that PmeR is the local repressor of the mexAB-oprM promoter and is able to regulate its own expression. The mechanism for this regulation includes binding to a pseudopalindromic operator site which overlaps both mexAB-oprM and pmeR promoters. We have also proven that flavonoids are able to interact with PmeR and induce a conformational change that interferes with the DNA binding ability of PmeR, thereby modulating mexAB-oprM and pmeR expression. Finally, we demonstrate by in vivo experiments that the PmeR/MexAB-OprM system contributes to the colonization of tomato plants. These results provide new insight into a transcriptional regulator and a transport system that play essential roles in the ability of P. syringae pv. tomato DC3000 to resist the action of flavonoids produced by the host.

TtgV represses two different promoters by recognizing different sequences.

Expression of the multidrug efflux pump ttgDEF and ttgGHI operons is modulated in vivo mainly by the TtgV repressor. TtgV is a multidrug recognition repressor that exhibits a DNA binding domain with a long interaction helix comprising residues 47 to 64. The pattern of expression of the two pumps is different in Pseudomonas putida: in the absence of effectors, the promoter for the ttgD gene is silent, whereas the ttgG gene is expressed at a high basal level. This correlates with the fact that TtgV exhibits a higher affinity for the ttgD operator (K(D)=10+/-1 nM) than for the ttgG (K(D)=19+/-1 nM) operator. Sequence analysis revealed that both operators are 40% identical, and mutational analysis of the ttgD and ttgG operators combined with electrophoretic mobility shift assays and in vivo expression analysis suggests that TtgV recognizes an inverted repeat with a high degree of palindromicity around the central axis. We generated a collection of alanine substitution mutants with substitutions between residues 47 and 64 of TtgV. The results of extensive combinations of promoter variants with these TtgV alanine substitution mutants revealed that TtgV modulates expression from ttgD and ttgG promoters through the recognition of both common and different sequences in the two promoters. In this regard, we found that TtgV mutants at residues 48, 50, 53, 54, 60, and 61 failed to bind ttgG but recognized the ttgD operator. TtgV residues R47, R52, L57, and T49 are critical for binding to both operators. Based on three-dimensional models, we propose that these residues contact nucleotides within the major groove of DNA.