Biological control of soil-borne pathogens by fluorescent pseudomonads.

Particular bacterial strains in certain natural environments prevent infectious diseases of plant roots. How these bacteria achieve this protection from pathogenic fungi has been analysed in detail in biocontrol strains of fluorescent pseudomonads. During root colonization, these bacteria produce antifungal antibiotics, elicit induced systemic resistance in the host plant or interfere specifically with fungal pathogenicity factors. Before engaging in these activities, biocontrol bacteria go through several regulatory processes at the transcriptional and post-transcriptional levels.

Persistence and cell culturability of biocontrol strain Pseudomonas fluorescens CHA0 under plough pan conditions in soil and influence of the anaerobic regulator gene anr.

Certain fluorescent pseudomonads can protect plants from soil-borne pathogens, and it is important to understand how these biocontrol agents survive in soil. The persistence of the biocontrol strain Pseudomonas fluorescens CHA0-Rif under plough pan conditions was assessed in non-sterile soil microcosms by counting total cells (immunofluorescence microscopy), intact cells (BacLight membrane permeability test), viable cells (Kogure's substrate-responsiveness test) and culturable cells (colony counts on selective plates) of the inoculant. Viable but non-culturable cells of CHA0-Rif (106 cells g-1 soil) were found in flooded microcosms amended with fermentable organic matter, in which the soil redox potential was low (plough pan conditions), in agreement with previous observations of plough pan samples from a field inoculated with CHA0-Rif. However, viable but non-culturable cells were not found in unamended flooded, amended unflooded or unamended unflooded (i.e. control) microcosms, suggesting that such cells resulted from exposure of CHA0-Rif to a combination of low redox potential and oxygen limitation in soil. CHA0-Rif is strictly aerobic. Its anaerobic regulator ANR is activated by low oxygen concentrations and it controls production of the biocontrol metabolite hydrogen cyanide under microaerophilic conditions. Under plough pan conditions, an anr-deficient mutant of CHA0-Rif and its complemented derivative displayed the same persistence pattern as CHA0-Rif, indicating that anr was not implicated in the formation of viable but non-culturable cells of this strain at the plough pan.

Gut physiology mediates a trade-off between adaptation to malnutrition and susceptibility to food-borne pathogens.

The animal gut plays a central role in tackling two common ecological challenges, nutrient shortage and food-borne parasites, the former by efficient digestion and nutrient absorption, the latter by acting as an immune organ and a barrier. It remains unknown whether these functions can be independently optimised by evolution, or whether they interfere with each other. We report that Drosophila melanogaster populations adapted during 160 generations of experimental evolution to chronic larval malnutrition became more susceptible to intestinal infection with the opportunistic bacterial pathogen Pseudomonas entomophila. However, they do not show suppressed immune response or higher bacterial loads. Rather, their increased susceptibility to P. entomophila is largely mediated by an elevated predisposition to loss of intestinal barrier integrity upon infection. These results may reflect a trade-off between the efficiency of nutrient extraction from poor food and the protective function of the gut, in particular its tolerance to pathogen-induced damage.

Use of fluorescent proteins to monitor the regulation of antifungal activity in root-associated Pseudomonas fluorescens CHA0

SUMMARY Roots of crop plants are the target of soil-borne pathogens, mainly fungi that cause considerable damage to plant health. By antagonizing these pathogens, some root-colonizing pseudomonads provide plants with efficient biological protection from disease. Pseudomonas fluorescens CHAO is a soil bacterium with the ability to suppress a considerable range of root diseases. A major characteristic conferring biocontrol capacity to this strain is the production of antifungal compounds, in particular 2,4-diacetyphloroglucinol (DAPG) and pyoluteorin (PLT). The regulation of the biosyntheses of these metabolites is complex and involves several regulatory systems responding to multiple environmental signals. In the present work, we have developed reporter systems based on green (GFP) and red fluorescent (DsRed) proteins to monitor regulation of antifungal gene expression in vitro and on plant roots. Stable and unstable GFP-based reporter fusions to the DAPG and PLT biosynthetic genes allowed us to demonstrate that P. fluorescens CHAO keeps the two antifungal compounds at a fine-tuned balance that can be affected by environmental signals. A GFP-based screening technique helped us to identify two novel regulators of balanced antibiotic production, i.e. MvaT and MvaV that are functionally and structurally related to the nucleoid-binding protein H-NS. They act in concert as global regulators of DAPG and PLT production and other biocontrol-related traits in P. fluorescens CHAO, and are essential for the bacterium's capacity to control a root disease caused by Pythium. The combined use of autofluorescent reporters, flow cytometry, and epifluorescence microscopy permitted us to visualize and quantify the expression of DAPG and PLT biosynthetic genes on roots. A GFP- and DsRed-based two-color approach was then developed to further improve the sensitivity of the flow cytometric quantitation method. The findings of this study shed more light on the complex regulatory mechanisms controlling antifungal activity of P. filuorescens in the rhizosphere. RESUME 4 e Les racines de plantes de culture sont la cible de divers pathogènes, principalement des champignons, qui nuisent gravement à la santé des plantes. Certains pseudomonades colonisant les racines peuvent avoir un effet antagoniste sur les pathogènes et protéger ainsi les plantes de manière efficace. Pseudomonas fluorescens CHAO est une bactérie du sol ayant la capacité de supprimer une gamme considérable de maladies racinaires. Une des caractéristiques principales conférant la capacité de biocontrôle à cette souche, est la production de composés antifongiques, en particulier le 2,4-diacétyphloroglucinol (DAPG) et la pyolutéorine (PLT). La régulation de la biosynthèse de ces métabolites est complexe et implique plusieurs systèmes régulateurs répondant à de multiples signaux environnementaux. Dans ce travail, nous avons développé des systèmes rapporteurs basés sur des protéines fluorescentes verte (GFP) et rouge (DsRed), afin d'étudier la régulation de l'expression des gènes d'antifongiques in vitro et sur les racines des plantes. Des fusions GFP stables et instables rapportrices de l'expression des gènes de biosynthèse du DAPG et de la PLT nous ont permis de démontrer que P. fluorescens CHAO gère les deux antifongiques dans une balance finement régulée pouvant être affectée par des signaux environnementaux. Une technique de criblage basée sur la GFP nous a permis d'identifier deux nouveaux régulateurs de la production d'antibiotiques, MvaT et MvaV, apparentés à la protéine H-NS liant l'ADN, Elles agissent de concert en tant que régulateurs globaux sur la production de DAPG et de PLT, ainsi que sur d'autres éléments relatifs au biocontrôle chez P. fluorescens CHAO. De plus, elles sont essentielles à la bactérie pour contrôler une maladie racinaire causée par Pythium. L'utilisation combinée de rapporteurs autofluorescents, de cytométrie de flux et de microscopie à épifluorescence nous a permis de visualiser et de quantifier l'expression des gènes de biosynthèse du DAPG et de la PLT sur les racines. Une approche utilisant simultanément la GFP et la DsRed a ensuite été développée afin d'améliorer la sensibilité de la méthode de quantification par cytométrie de flux. Les résultats de cette étude ont apporté plus de lumière sur les mécanismes régulateurs complexes contrôlant l'activité antifongique de P. fluorescens dans la rizosphère.

The endodermis.

A Casparian strip-bearing endodermis is a feature that has been invariably present in the roots of ferns and angiosperms for approximately 400 million years. As the innermost cortical layer that surrounds the central vasculature of roots, the endodermis acts as a barrier to the free diffusion of solutes from the soil into the stele. Based on an enormous body of anatomical and physiological work, the protective endodermal diffusion barrier is thought to be of major importance for many aspects of root biology, reaching from efficient water and nutrient transport to defense against soil-borne pathogens. Until recently, however, we were ignorant about the genes and mechanisms that drive the differentiation of this intricately structured barrier. Recent work in Arabidopsis has now identified the first major players in Casparian strip formation. A mechanistic understanding of endodermal differentiation will finally allow us to specifically interfere with endodermal barrier function and study the effects on plant growth and survival under various stress conditions. Here, I critically review the major findings and models related to endodermal structure and function from other plant species and assess them in light of recent molecular data from Arabidopsis, pointing out where the older, descriptive work can provide a framework and inspiration for further molecular dissection.

Evolutionary patchwork of an insecticidal toxin shared between plant-associated pseudomonads and the insect pathogens Photorhabdus and Xenorhabdus.

BACKGROUND: Root-colonizing fluorescent pseudomonads are known for their excellent abilities to protect plants against soil-borne fungal pathogens. Some of these bacteria produce an insecticidal toxin (Fit) suggesting that they may exploit insect hosts as a secondary niche. However, the ecological relevance of insect toxicity and the mechanisms driving the evolution of toxin production remain puzzling. RESULTS: Screening a large collection of plant-associated pseudomonads for insecticidal activity and presence of the Fit toxin revealed that Fit is highly indicative of insecticidal activity and predicts that Pseudomonas protegens and P. chlororaphis are exclusive Fit producers. A comparative evolutionary analysis of Fit toxin-producing Pseudomonas including the insect-pathogenic bacteria Photorhabdus and Xenorhadus, which produce the Fit related Mcf toxin, showed that fit genes are part of a dynamic genomic region with substantial presence/absence polymorphism and local variation in GC base composition. The patchy distribution and phylogenetic incongruence of fit genes indicate that the Fit cluster evolved via horizontal transfer, followed by functional integration of vertically transmitted genes, generating a unique Pseudomonas-specific insect toxin cluster. CONCLUSIONS: Our findings suggest that multiple independent evolutionary events led to formation of at least three versions of the Mcf/Fit toxin highlighting the dynamic nature of insect toxin evolution.

Promise for plant pest control: root-associated pseudomonads with insecticidal activities.

Insects are an important and probably the most challenging pest to control in agriculture, in particular when they feed on belowground parts of plants. The application of synthetic pesticides is problematic owing to side effects on the environment, concerns for public health and the rapid development of resistance. Entomopathogenic bacteria, notably Bacillus thuringiensis and Photorhabdus/Xenorhabdus species, are promising alternatives to chemical insecticides, for they are able to efficiently kill insects and are considered to be environmentally sound and harmless to mammals. However, they have the handicap of showing limited environmental persistence or of depending on a nematode vector for insect infection. Intriguingly, certain strains of plant root-colonizing Pseudomonas bacteria display insect pathogenicity and thus could be formulated to extend the present range of bioinsecticides for protection of plants against root-feeding insects. These entomopathogenic pseudomonads belong to a group of plant-beneficial rhizobacteria that have the remarkable ability to suppress soil-borne plant pathogens, promote plant growth, and induce systemic plant defenses. Here we review for the first time the current knowledge about the occurrence and the molecular basis of insecticidal activity in pseudomonads with an emphasis on plant-beneficial and prominent pathogenic species. We discuss how this fascinating Pseudomonas trait may be exploited for novel root-based approaches to insect control in an integrated pest management framework.

Regulation of the expression of Fit insect toxin locus genes in the root-associated biocontrol pseudomonad CHA0

The root-colonizing Pseudomonas fluorescens strain CHA0 is a biocontrol agent of soil-borne plant diseases caused by fungal and oomycete pathogens. Remarkably, this plant-beneficial pseudomonad is also endowed with potent insecticidal activity that depends on the production of a large protein toxin termed Fit (for P. fluorescens insecticidal toxin). In our present work, the genomic locus encoding the P. fluorescens insect toxin is subjected to a detailed molecular analysis. The Fit toxin gene fitD is flanked upstream by the fitABC genes and downstream by the fitE gene that encode the ABC transporter, membrane fusion, and outer membrane efflux components of a type I protein secretion system predicted to function in toxin export. The fitF, fitG, and fitH genes located downstream of fitE code for regulatory proteins having domain structures typical of signal transduction histidine kinases, LysR-type transcriptional regulators, and response regulators, respectively. The role of these insect toxin locus-associated control elements is being investigated with mutants defective for the regulatory genes and with GFP-based reporter fusions to putative promoter regions upstream of the transporter genes fitA and fitE, the toxin gene fitD, and the regulatory genes fitF and fitH. Our preliminary findings suggest that the three regulators interact with known global regulators of biocontrol factor expression to control Fit toxin expression and secretion.

Tn5-directed cloning of pqq genes from Pseudomonas fluorescens CHA0: mutational inactivation of the genes results in overproduction of the antibiotic pyoluteorin.

Pseudomonas fluorescens CHA0 produces several secondary metabolites, e.g., the antibiotics pyoluteorin (Plt) and 2,4-diacetylphloroglucinol (Phl), which are important for the suppression of root diseases caused by soil-borne fungal pathogens. A Tn5 insertion mutant of strain CHA0, CHA625, does not produce Phl, shows enhanced Plt production on malt agar, and has lost part of the ability to suppress black root rot in tobacco plants and take-all in wheat. We used a rapid, two-step cloning-out procedure for isolating the wild-type genes corresponding to those inactivated by the Tn5 insertion in strain CHA625. This cloning method should be widely applicable to bacterial genes tagged with Tn5. The region cloned from P. fluorescens contained three complete open reading frames. The deduced gene products, designated PqqFAB, showed extensive similarities to proteins involved in the biosynthesis of pyrroloquinoline quinone (PQQ) in Klebsiella pneumoniae, Acinetobacter calcoaceticus, and Methylobacterium extorquens. PQQ-negative mutants of strain CHA0 were constructed by gene replacement. They lacked glucose dehydrogenase activity, could not utilize ethanol as a carbon source, and showed a strongly enhanced production of Plt on malt agar. These effects were all reversed by complementation with pqq+ recombinant plasmids. The growth of a pqqF mutant on ethanol and normal Plt production were restored by the addition of 16 nM PQQ. However, the Phl- phenotype of strain CHA625 was due not to the pqq defect but presumably to a secondary mutation. In conclusion, a lack of PQQ markedly stimulates the production of Plt in P. fluorescens.

Molecular basis and regulation of insect pathogenicity in plant-beneficial pseudomonads

Les bactéries du genre Pseudomonas ont la capacité étonnante de s'adapter à différents habitats et d'y survivre, ce qui leur a permis de conquérir un large éventail de niches écologiques et d'interagir avec différents organismes hôte. Les espèces du groupe Pseudomonas fluorescens peuvent être facilement isolées de la rhizosphère et sont communément connues comme des Pseudomonas bénéfiques pour les plantes. Elles sont capables d'induire la résistance systémique des plantes, d'induire leur croissance et de contrer des phytopathogènes du sol. Un sous-groupe de ces Pseudomonas a de plus développé la capacité d'infecter et de tuer certaines espèces d'insectes. Approfondir les connaissances sur l'interaction de ces bactéries avec les insectes pourraient conduire au développement de nouveaux biopesticides pour la protection des cultures. Le but de cette thèse est donc de mieux comprendre la base moléculaire, l'évolution et la régulation de la pathogénicité des Pseudomonas plante-bénéfiques envers les insectes. Plus spécifiquement, ce travail a été orienté sur l'étude de la production de la toxine insecticide appelée Fit et sur l'indentification d'autres facteurs de virulence participant à la toxicité de la bactérie envers les insectes. Dans la première partie de ce travail, la régulation de la production de la toxine Fit a été évaluée par microscopie à épifluorescence en utilisant des souches rapportrices de Pseudomonas protegens CHA0 qui expriment la toxine insecticide fusionnée à une protéine fluorescente rouge, au site natif du gène de la toxine. Celle-ci a été détectée uniquement dans l'hémolymphe des insectes et pas sur les racines des plantes, ni dans les milieux de laboratoire standards, indiquant une production dépendante de l'hôte. L'activation de la production de la toxine est contrôlée par trois protéines régulatrices dont l'histidine kinase FitF, essentielle pour un contrôle précis de l'expression et possédant un domaine "senseur" similaire à celui de la kinase DctB qui régule l'absorption de carbone chez les Protéobactéries. Il est donc probable que, durant l'évolution de FitF, un réarrangement de ce domaine "senseur" largement répandu ait contribué à une production hôte-spécifique de la toxine. Les résultats de cette étude suggèrent aussi que l'expression de la toxine Fit est plutôt réprimée en présence de composés dérivés des plantes qu'induite par la perception d'un signal d'insecte spécifique. Dans la deuxième partie de ce travail, des souches mutantes ciblant des facteurs de virulence importants identifiés dans des pathogènes connus ont été générées, dans le but d'identifier ceux avec une virulence envers les insectes atténuée. Les résultats ont suggéré que l'antigène O du lipopolysaccharide (LPS) et le système régulateur à deux composantes PhoP/PhoQ contribuent significativement à la virulence de P. protegens CHA0. La base génétique de la biosynthèse de l'antigène O dans les Pseudomonas plante-bénéfiques et avec une activité insecticide a été élucidée et a révélé des différences considérables entre les lignées suite à des pertes de gènes ou des acquisitions de gènes par transfert horizontal durant l'évolution de certaines souches. Les chaînes latérales du LPS ont été montrées comme vitales pour une infection des insectes réussie par la souche CHA0, après ingestion ou injection. Les Pseudomonas plante-bénéfiques, avec une activité insecticide sont naturellement résistants à la polymyxine B, un peptide antimicrobien modèle. La protection contre ce composé antimicrobien particulier dépend de la présence de l'antigène O et de la modification du lipide A, une partie du LPS, avec du 4-aminoarabinose. Comme les peptides antimicrobiens cationiques jouent un rôle important dans le système immunitaire des insectes, l'antigène O pourrait être important chez les Pseudomonas insecticides pour surmonter les mécanismes de défense de l'hôte. Le système PhoP/PhoQ, connu pour contrôler les modifications du lipide A chez plusieurs bactéries pathogènes, a été identifié chez Pseudomonas chlororaphis PCL1391 et P. protegens CHA0. Pour l'instant, il n'y a pas d'évidence que des modifications du lipide A contribuent à la pathogénicité de cette bactérie envers les insectes. Cependant, le senseur-kinase PhoQ est requis pour une virulence optimale de la souche CHA0, ce qui suggère qu'il régule aussi l'expression des facteurs de virulence de cette bactérie. Les découvertes de cette thèse démontrent que certains Pseudomonas associés aux plantes sont de véritables pathogènes d'insectes et donnent quelques indices sur l'évolution de ces microbes pour survivre dans l'insecte-hôte et éventuellement le tuer. Les résultats suggèrent également qu'une recherche plus approfondie est nécessaire pour comprendre comment ces bactéries sont capables de contourner ou surmonter la réponse immunitaire de l'hôte et de briser les barrières physiques pour envahir l'insecte lors d'une infection orale. Pour cela, les futures études ne devraient pas uniquement se concentrer sur le côté bactérien de l'interaction hôte-microbe, mais aussi étudier l'infection du point de vue de l'hôte. Les connaissances gagnées sur la pathogénicité envers les insectes des Pseudomonas plante-bénéfiques donnent un espoir pour une future application en agriculture, pour protéger les plantes, non seulement contre les maladies, mais aussi contre les insectes ravageurs. -- Pseudomonas bacteria have the astonishing ability to survive within and adapt to different habitats, which has allowed them to conquer a wide range of ecological niches and to interact with different host organisms. Species of the Pseudomonas fluorescens group can readily be isolated from plant roots and are commonly known as plant-beneficial pseudomonads. They are capable of promoting plant growth, inducing systemic resistance in the plant host and antagonizing soil-borne phytopathogens. A defined subgroup of these pseudomonads evolved in addition the ability to infect and kill certain insect species. Profound knowledge about the interaction of these particular bacteria with insects could lead to the development of novel biopesticides for crop protection. This thesis thus aimed at a better understanding of the molecular basis, evolution and regulation of insect pathogenicity in plant-beneficial pseudomonads. More specifically, it was outlined to investigate the production of an insecticidal toxin termed Fit and to identify additional factors contributing to the entomopathogenicity of the bacteria. In the first part of this work, the regulation of Fit toxin production was probed by epifluorescence microscopy using reporter strains of Pseudomonas protegens CHAO that express a fusion between the insecticidal toxin and a red fluorescent protein in place of the native toxin gene. The bacterium was found to express its insecticidal toxin only in insect hemolymph but not on plant roots or in common laboratory media. The host-dependent activation of Fit toxin production is controlled by three local regulatory proteins. The histidine kinase of this regulatory system, FitF, is essential for the tight control of toxin expression and shares a sensing domain with DctB, a sensor kinase regulating carbon uptake in Proteobacteria. It is therefore likely that shuffling of a ubiquitous sensor domain during the evolution of FitF contributed to host- specific production of the Fit toxin. Findings of this study additionally suggest that host-specific expression of the Fit toxin is mainly achieved by repression in the presence of plant-derived compounds rather than by induction upon perceiving an insect-specific signal molecule. In the second part of this thesis, mutant strains were generated that lack factors previously shown to be important for virulence in prominent pathogens. A screening for attenuation in insect virulence suggested that lipopolysaccharide (LPS) O-antigen and the PhoP-PhoQ two-component regulatory system significantly contribute to virulence of P. protegens CHAO. The genetic basis of O-antigen biosynthesis in plant-beneficial pseudomonads displaying insect pathogenicity was elucidated and revealed extensive differences between lineages due to reduction and horizontal acquisition of gene clusters during the evolution of several strains. Specific 0 side chains of LPS were found to be vital for strain CHAO to successfully infect insects by ingestion or upon injection. Insecticidal pseudomonads with plant-beneficial properties were observed to be naturally resistant to polymyxin B, a model antimicrobial peptide. Protection against this particular antimicrobial compound was dependent on the presence of O-antigen and modification of the lipid A portion of LPS with 4-aminoarabinose. Since cationic antimicrobial peptides play a major role in the immune system of insects, O-antigenic polysaccharides could be important for insecticidal pseudomonads to overcome host defense mechanisms. The PhoP-PhoQ system, which is well-known to control lipid A modifications in several pathogenic bacteria, was identified in Pseudomonas chlororaphis PCL1391 and P. protegens CHAO. No evidence was found so far that lipid A modifications contribute to insect pathogenicity in this bacterium. However, the sensor kinase PhoQ was required for full virulence of strain CHAO suggesting that it additionally regulates the expression of virulence factors in this bacterium. The findings of this thesis demonstrate that certain plant-associated pseudomonads are true insect pathogens and give some insights into how these microbes evolved to survive within and eventually kill the insect host. Results however also point out that more in-depth research is needed to know how exactly these fascinating bacteria manage to bypass or overcome host immune responses and to breach physical barriers to invade insects upon oral infection. To achieve this, future studies should not only focus on the bacterial side of the microbe-host interactions but also investigate the infection from a host-oriented view. The knowledge gained about the entomopathogenicity of plant-beneficial pseudomonads gives hope for their future application in agriculture to protect plants not only against plant diseases but also against insect pests.

Potential exposure to hepatitis C virus through accidental blood contact in interventional radiology.

PURPOSE: To quantify the prevalence of accidental blood exposure (ABE) among interventional radiologists and contrast that with the prevalence of patients with hepatitis C virus (HCV) undergoing interventional radiology procedures. MATERIALS AND METHODS: A multicenter epidemiologic study was conducted in radiology wards in France. The risk of ABE to radiologists was assessed based on personal interviews that determined the frequency and type of ABE and the use of standard protective barriers. Patients who underwent invasive procedures underwent prospective sampling for HCV serologic analysis. HCV viremia was measured in patients who tested positive for HCV. RESULTS: Of the 77 radiologists who participated in 11 interventional radiology wards, 44% reported at least one incident of mucous membrane blood exposure and 52% reported at least one percutaneous injury since the beginning of their occupational activity. Compliance with standard precautions was poor, especially for the use of protective clothes and safety material. Overall, 91 of 944 treated patients (9.7%) tested positive for HCV during the study period, of whom 90.1% had positive viremia results, demonstrating a high potential for contamination through blood contacts. CONCLUSIONS: The probability of HCV transmission from contact with contaminated blood after percutaneous injury ranged from 0.013 to 0.030; the high frequency of accidental blood exposure and high percentage of patients with HCV could generate a risk of exposure to HCV for radiologists who perform invasive procedures with frequent blood contact. The need to reinforce compliance with standard hygiene precautions is becoming crucial for medical and technical personnel working in these wards.

Control and host-dependent activation of insect toxin expression in a root-associated biocontrol pseudomonad.

Pseudomonas fluorescens CHA0 is a root-associated biocontrol agent that suppresses soil-borne fungal diseases of crops. Remarkably, the pseudomonad is also endowed with systemic and oral activity against pest insects which depends on the production of the insecticidal Fit toxin. The toxin gene (fitD) is part of a virulence cassette encoding three regulators (FitF, FitG, FitH) and a type I secretion system (FitABC-E). Immunoassays with a toxin-specific antibody and transcriptional analyses involving fitG and fitH deletion and overexpression mutants identified LysR family regulator FitG and response regulator FitH as activator and repressor, respectively, of Fit toxin and transporter expression. To visualize and quantify toxin expression in single live cells by fluorescence microscopy, we developed reporters which in lieu of the native toxin protein express a fusion of the Fit toxin with red fluorescent mCherry. In a wild-type background, expression of the mCherry-tagged Fit toxin was activated at high levels in insect hosts, i.e. when needed, yet not on plant roots or in batch culture. By contrast, a derepressed fitH mutant expressed the toxin in all conditions. P. fluorescens hence can actively induce insect toxin production in response to the host environment, and FitH and FitG are key regulators in this mechanism.

Inactivation des pathogènes des produits sanguins labiles: entre enjeux financiers et conséquences possibles à long terme [Pathogen reduction of blood components: from financial issues to possible long-term consequences].

Pathogen inactivation of blood products represents a global and major paradigm shift in transfusion medicine. In the next near future, it is likely that most blood products will be inactivated by various physicochemical approaches. The concept of blood safety will be challenged as well as transfusion medicine practice, notably for donor selection or biological qualification. In this context, it seems mandatory to develop analytical economic approaches by assessing costs-benefits ratio of blood transfusion as well as to set up cohorts of patients based on hemovigilance networks allowing rigorous scientific analysis of the benefits and the risks of blood transfusion at short- and long-term.

Use of green fluorescent protein-based reporters to monitor balanced production of antifungal compounds in the biocontrol agent Pseudomonas fluorescens CHA0.

AIMS: To develop reporter constructs based on stable and unstable variants of the green fluorescent protein (GFP) for monitoring balanced production of antifungal compounds that are crucial for the capacity of the root-colonizing Pseudomonas fluorescens strain CHA0 to control plant diseases caused by soil-borne pathogenic fungi. METHODS AND RESULTS: Pseudomonas fluorescens CHA0 produces the three antifungal metabolites 2,4-diacetylphloroglucinol (DAPG), pyoluteorin (PLT) and pyrrolnitrin (PRN). The gfp[mut3] and gfp[AAV] reporter genes were fused to the promoter regions of the DAPG, PLT and PRN biosynthetic genes. The reporter fusions were then used to follow the kinetics of expression of the three antifungal metabolites in a microplate assay. DAPG and PLT were found to display an inverse relationship in which each metabolite activates its own biosynthesis while repressing the synthesis of the other metabolite. PRN appears not to be involved in this balance. However, the microbial and plant phenolic metabolite salicylate was found to interfere with the expression of both DAPG and PLT. CONCLUSIONS: The results obtained provide evidence that P. fluorescens CHA0 may keep the antifungal compounds DAPG and PLT at a fine-tuned balance that can be affected by certain microbial and plant phenolics. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, the present study is the first to use stable and unstable GFP variants to study antibiotic gene expression in a biocontrol pseudomonad. The developed reporter fusions will be a highly valuable tool to study in situ expression of this bacterial biocontrol trait on plant roots, i.e. at the site of pathogen suppression.