Pyoluteorin production by Pseudomonas fluorescens strain CHA0 is involved in the suppression of Pythium damping-off of cress but not of cucumber

Pseudomonas fluorescens strain CHA0 is an effective biocontrol agent of various soilborne pathogens. It controls damping-off or root rot caused byPythium ultimum on cucumber, wheat and cress. Strain CHA0 synthesizes several antibiotic metabolites such as hydrogen cyanide, 2,4-diacetylphloroglucinol, and pyoluteorin. The role of pyoluteorin in the suppression of damping-off was investigated. Two Tn5 mutants (CHA660 and CHA661) of strain CHA0 were isolated which had lost the capacity to produce pyoluteorin but still produced 2,4-diacteylphloroglucinol and HCN. These mutants still inhibitedP. ultimum on malt agar (which favours the production of 2,4-diacetylphloroglucinol) but had partially lost the ability to inhibit this pathogen on King's B agar (which favours the production of pyoluteorin). The two pyoluteorin-negative mutants showed a reduced capacity to suppress damping-off of cress caused byP. ultimum but were as effective in the protection of cucumber against this pathogen as the wild-type strain. These results indicate that, depending on the plant, pyoluteorin production plays a role in the suppression of damping-off by strain CHA0 without being a major mechanism in disease suppression. We suggest that the contribution of pyoluteorin to the biocontrol activity of strain CHA0 is determined by the quantity of this antibiotic produced in the rhizosphere, which might depend on the root exudates of the host plant.

Enhanced production of indole-3-acetic acid by a genetically modified strain of Pseudomonas fluorescens CHA0 affects root growth of cucumber but does not improve protection of the plant against Pythium root rot

The biocontrol strain CHA0 of Pseudomonas fluorescens produces small amounts of indole-3-acetic acid via the tryptophan side chain oxidase and the tryptophan transaminase pathways. A recombinant plasmid (pME3468) expressing the tryptophan monooxygenase pathway was introduced into strain CHA0; this resulted in elevated synthesis of indole-3-acetic acid in vitro, especially after addition of -tryptophan. In natural soil, strain CHA0/pME3468 increased fresh root weight of cucumber by 17-36%, compared to the effect of strain CHA0; root colonization was about 106 cells per g of root. However, both strains gave similar protection of cucumber against Pythium ultimum. In autoclaved soil, at 6×107 cells per g of root, strain CHA0 stimulated growth of roots and shoots, whereas strain CHA0/pME3468 caused root stunting and strong reduction of plant weight. These results are in agreement with the known effects of exogenous indole-3-acetic acid on plant roots and suggest that in the system examined, indole-3-acetic acid does not contribute to the biocontrol properties of strain CHA0.

Intracellular excision and reintegration dynamics of the ICEclc genomic island of Pseudomonas knackmussii sp. strain B13.

Genomic islands are DNA elements acquired by horizontal gene transfer that are common to a large number of bacterial genomes, which can contribute specific adaptive functions, e.g. virulence, metabolic capacities or antibiotic resistances. Some genomic islands are still self-transferable and display an intricate life-style, reminiscent of both bacteriophages and conjugative plasmids. Here we studied the dynamical process of genomic island excision and intracellular reintegration using the integrative and conjugative element ICEclc from Pseudomonas knackmussii B13 as model. By using self-transfer of ICEclc from strain B13 to Pseudomonas putida and Cupriavidus necator as recipients, we show that ICEclc can target a number of different tRNA(Gly) genes in a bacterial genome, but only those which carry the GCC anticodon. Two conditional traps were designed for ICEclc based on the attR sequence, and we could show that ICEclc will insert with different frequencies in such traps producing brightly fluorescent cells. Starting from clonal primary transconjugants we demonstrate that ICEclc is excising and reintegrating at detectable frequencies, even in the absence of recipient. Recombination site analysis provided evidence to explain the characteristics of a larger number of genomic island insertions observed in a variety of strains, including Bordetella petri, Pseudomonas aeruginosa and Burkholderia.

Plastic and evolutionary responses of cell size and number to larval malnutrition in Drosophila melanogaster.

Both development and evolution under chronic malnutrition lead to reduced adult size in Drosophila. We studied the contribution of changes in size vs. number of epidermal cells to plastic and evolutionary reduction of wing size in response to poor larval food. We used flies from six populations selected for tolerance to larval malnutrition and from six unselected control populations, raised either under standard conditions or under larval malnutrition. In the control populations, phenotypic plasticity of wing size was mediated by both cell size and cell number. In contrast, evolutionary change in wing size, which was only observed as a correlated response expressed on standard food, was mediated entirely by reduction in cell number. Plasticity of cell number had been lost in the selected populations, and cell number did not differ between the sexes despite males having smaller wings. Results of this and other experimental evolution studies are consistent with the hypothesis that alleles which increase body size through prolonged growth affect wing size mostly via cell number, whereas alleles which increase size through higher growth rate do so via cell size.

Construction and analysis of a new conditional ferritin H knockout mouse strain/

Résumé Le fer joue un rôle important dans la plupart des fonctions biologiques mais sa présence excessive provoque la production de molécules réactives d'oxygène (ROS) qui peuvent contribuer à diverses maladies. La protéine de stockage du fer, la ferritine H, capte l'excès en fer et le stocke sous forme non-toxique, ce qui empêche des dommages potentiels. La délétion de la ferritine H dans des souris knock-out a été essayée antérieurement, mais ces souris mouraient au stade précoce du développement embryonnaire. Pour étudier l'importance du fer, et en particulier son stockage dans la ferritine, et pour pouvoir mieux comprendre les fonctions de la ferritine H, nous avons créé un modèle de souris knock-out conditionnelles de la ferritine H, selon le système classique de Cre-LoxP. Le premier exon et la région du promoteur du gène de la ferritine H ont été entourés de sites loxP. La mortalité embryonnaire provoquée par la délétion constitutive du gène de la ferritine H a été confirmée en croisant nos souris avec des souris exprimant nestin-Cre1. En croisant nos souris avec des souris transgéniques Mx-Cre, nous avons observé que l'induction de Cre par injection de polyI-polyC provoque la délétion presque complète de la ferritine H dans le foie (> 99%) et la rate (> 88%). Ces tissus ont également perdu une grande partie de leur réserve de fer. Cette observation apporte pour la première fois la preuve in vivo que la ferritine H est indispensable pour le stockage du fer, que les fonctions de la ferritine H et de la ferritine L ne sont pas équivalentes, et que la ferritine L ne peut pas assumer seule la fonction de stockage du fer. Dans le foie des souris knock-out, l'expression de l'ARN messager de l'hepcidine a été induite après 10 jours. En même temps, l'expression de l'ARN messager des gènes codant pour des protéines de l'absorption de fer (DMT1, ferroportin, Dcytb1 et hephaestin) a été réprimée mais dans le duodénum seulement. L'expression d'hepcidine est inversément corrélée avec celle des gènes liés à l'absorption de fer. Cette observation corrobore des études antérieures. Mais, en plus, elle montre également que cette répression se produit seulement dans l'intestin. Nous pouvons ainsi tirer la conclusion suivante : ou bien l'hepcidine a un récepteur spécifique dans le duodénum ou bien les gènes liés à l'absorption de fer dans le duodénum ont un facteur spécifique de transcription sensible à l'hepcidine. Aucune répression de DMT1 et de ferroportin n'a été observée dans les macrophages de la rate après l'induction d'hepcidine. La délétion de ferritine H a entraîné une augmentation du taux de mortalité des cellules hépatiques, ainsi que des altérations dans l'architecture normale du tissu de la rate. Vu par l'immunohistologie, le nombre de lymphocytes B et T était réduit dans la rate, tendant à démontrer que la ferritine H et l'homéostase du fer jouent un rôle dans l'immunité. En conclusion, le modèle de souris knock-out conditionnelles de la ferritine H nous fournit un outil précieux pour l'étude in vivo du rôle joué par la ferritine dans l'homéostase du fer, dans les dommages créés par les ROS, ainsi que dans l'apoptose et l'immunité. Summary Iron plays an important role in most biological functions. However, excess of iron results in production of reactive oxygen species (ROS) which could substantially contribute to pathology of various diseases. Ferritin H scavenges excess of iron and stores it in non-toxic form and potentially prevents the damage. Fenitin H targeting in mice has been attempted before, however, straight knockout was lethal in early embryonic stage. To study the role of iron and its storage protein ferritin and to further elucidate ferritin H functions, we aimed at creating a conditional ferritin H knockout mouse model by classical Cre-LoxP system. First exon along with promoter region of the ferritin H gene was foxed. Embryonic lethality of the constitutive ferritin H deletion was confirmed by crossing the foxed mice with mice expressing nestin Cre-1 as transgene. Almost complete deletion was observed in liver (> 99%) and spleen (>88%) upon induction of Cre by injecting polyI-polyC in Fth Lox/Lox; MxCre mice. These tissues also lost substantial fraction of their iron stores. This provides first in vivo evidence that ferritin H is required for iron storage, ferritin H and L functions are not redundant and that ferritin L cannot perform iron storage function alone. Hepcidin mRNA expression was induced after 10 days in the livers of deleted mice and, simultaneously, mRNA expression of iron absorption related genes (DMT 1, ferroportin, Dcytb1 and hephaestin) was repressed in duodenum only. Hepcidin expression is inversely correlated with that of duodenal iron absorption related genes. This is in agreement with previous studies. However, we also show that this repression happens only in intestine. This leads to the conclusion that either hepcidin has a specific receptor in duodenum or the iron absorption related genes have duodenum specific transcription factor that is responsive to hepcidin. No repression of DMT1 and ferroportin was observed in spleen macrophages upon hepcidin induction. Ferritin H deletion showed increased cell death in liver and disruption of normal architecture of spleen. B lymphocytes were reduced in spleen on immunohistology which point towards a role of ferritin H and iron homeostasis in immunity. In conclusion, ferritin H conditional knockout mouse model provides us with an invaluable tool to study the in vivo role of ferritin H in iron homeostasis, ROS mediated damage, apoptosis and immunity.

Cell-cell communication in the biocontrol strain Pseudomonas fluorescens CHA0

Summary Pseudomonas fluorescens CHAO is a soil bacterium which was isolated near Morens (Switzerland) and which protects plants from root-pathogenic fungi. This protection is due to extracellular secondary metabolites whose synthesis is regulated by the two-component system GacS/GacA in strain CHAO. Extracellular signals of bacterial origin activate this regulatory system. These signals are different from N-acyl-homoserine lactones, are extracted by dichloromethane and appear to have a low molecular weight. Preliminary evidence was obtained from a small molecule m/z 278 produced by strain CHAO. Similar signals capable of activating GacS/GacA-dependent regulation in strain CHAO were found in a large number of different Gram-negative bacteria. Once activated by signal(s), the sensor GacS is assumed to phosphorylate the response regulator GacA, which positively influences a regulatory cascade, resulting in the synthesis of secondary metabolites. This cascade includes three GacA-controlled small regulatory RNAs and two translational repressor proteins. The regulatory RNAs titrate the repressor proteins; this allows translation of target genes and the synthesis of exoenzymes and secondary metabolites such as antibiotics and hydrogen cyanide. A GFP-based sensor for signal detection was constructed in strain CHAO by fusing the gfp reporter gene to the rsmZ small RNA gene. CHAO mutants defective for signal production were isolated following transposon insertion mutagenesis. In one class of mutants obtained, the gacS gene was inactivated, indicating that GacS/GacA positively controls signal production. In a second class, the thiC gene required for thiamine (vitamin B1) biosynthesis was disrupted. Addition of excess (> 10E-6 M) thiamine to the medium restored signal production. By contrast, when the thiamine concentration was just sufficient to allow normal growth, no production of signal(s) was observed. The mechanism by which thiamine activates signal production remains to be elucidated. Résumé Pseudomonas fluorescens CHAO est une bactérie du sol, isolée près de Morens (Suisse), qui a la capacité de protéger les plantes contre des champignons pathogènes de la racine. Cette protection provient de métabolites secondaires excrétés par la bactérie, dont la synthèse est régulée par le système à deux composants GacS/GacA. Des signaux extracellulaires d'origine bactérienne activent ce système de régulation. Ces signaux, différents des N-acyl¬homosérines lactones, sont extraits par le dichlorométhane et semblent avoir une petite masse moléculaire. Une molécule (masse m/z 278) a été mise en évidence par des expériences préliminaires chez la souche CHAO. Des signaux similaires, capables d'activer la régulation dépendante de GacS/GacA chez la souche CHAO, ont été trouvés chez un grand nombre de bactéries à Gram négative. Une fois activé par le(s) signal(aux), le senseur GacS est supposé phosphoryler le régulateur de réponse GacA, qui influence positivement la cascade de régulation menant à la synthèse des métabolites secondaires. Cette cascade inclut trois petits ARNs régulateurs contrôlés par GacA et deux protéines répresseurs de la traduction. Les ARNs régulateurs titrent les protéines répresseurs, ce qui permet la traduction des gènes cibles et la synthèse d'exoenzymes et de métabolites secondaires tel les antibiotiques et le cyanure d'hydrogène. Un senseur basé sur la GFP pour la détection de signaux a été construit dans la souche CHAO en fusionnant le gène rapporteur gfp au gène de petit ARN rsmZ. Des mutants de CHAO déficients pour la production de signaux ont été isolés au moyen d'une mutagenèse par insertion de transposon. Chez une classe de mutants obtenus, le gène gacS a été inactivé, indiquant que GacS/GacA contrôle positivement la production de signaux. Dans une seconde classe, le gène thiC nécessaire à la biosynthèse de thiamine (vitamine B1) a été interrompu. L'addition en excès (> 10E-6 M) de thiamine au milieu restaure la production de signaux. A l'opposé, quand la concentration de thiamine est juste suffisante pour permettre une croissance normale, aucune production de signaux n'a été observée. Le mécanisme par lequel la thiamine active la production de signaux reste à élucider.

Strain-transcending Fc-dependent killing of Plasmodium falciparum by merozoite surface protein 2 allele-specific human antibodies.

It is widely accepted that antibody responses against the human parasitic pathogen Plasmodium falciparum protect the host from the rigors of severe malaria and death. However, there is a continuing need for the development of in vitro correlate assays of immune protection. To this end, the capacity of human monoclonal and polyclonal antibodies in eliciting phagocytosis and parasite growth inhibition via Fcγ receptor-dependent mechanisms was explored. In examining the extent to which sequence diversity in merozoite surface protein 2 (MSP2) results in the evasion of antibody responses, an unexpectedly high level of heterologous function was measured for allele-specific human antibodies. The dependence on Fcγ receptors for opsonic phagocytosis and monocyte-mediated antibody-dependent parasite inhibition was demonstrated by the mutation of the Fc domain of monoclonal antibodies against both MSP2 and a novel vaccine candidate, peptide 27 from the gene PFF0165c. The described flow cytometry-based functional assays are expected to be useful for assessing immunity in naturally infected and vaccinated individuals and for prioritizing among blood-stage antigens for inclusion in blood-stage vaccines.

Extracellular protease and phospholipase C are controlled by the global regulatory gene gacA in the biocontrol strain Pseudomonas fluorescens CHA0.

Pseudomonas fluorescens strain CHA0 protects plants from various root diseases. Antibiotic metabolites synthesized by this strain play an important role in disease suppression; their production is mediated by the global activator gene gacA. Here we show by complementation that the gacA gene is also essential for the expression of two extracellular enzymes in P. fluorescens CHA0: phospholipase C and a 47-kDa metalloprotease. In contrast, the production of another exoenzyme, lipase, is not regulated by the gacA gene. Protease, phospholipase and antibiotics of P. fluorescens are all known to be optimally produced at the end of exponential growth; thus, the gacA gene appears to be a general stationary-phase regulator.

Orthotropic active strain models for the numerical simulation of cardiac biomechanics

An active strain formulation for orthotropic constitutive laws arising in cardiac mechanics modeling is introduced and studied. The passive mechanical properties of the tissue are described by the Holzapfel-Ogden relation. In the active strain formulation, the Euler-Lagrange equations for minimizing the total energy are written in terms of active and passive deformation factors, where the active part is assumed to depend, at the cell level, on the electrodynamics and on the specific orientation of the cardiac cells. The well-posedness of the linear system derived from a generic Newton iteration of the original problem is analyzed and different mechanical activation functions are considered. In addition, the active strain formulation is compared with the classical active stress formulation from both numerical and modeling perspectives. Taylor-Hood and MINI finite elements are employed to discretize the mechanical problem. The results of several numerical experiments show that the proposed formulation is mathematically consistent and is able to represent the main key features of the phenomenon, while allowing savings in computational costs.

Strain diversity of mycobacteria isolated from pulmonary tuberculosis patients at Debre Birhan Hospital, Ethiopia.

BACKGROUND: Ethiopia ranks seventh in the list of 22 high tuberculosis (TB) burden countries, with an incidence rate of 379 cases per 100 000 population for TB all forms. However, information on the genomic diversity of Mycobacterium tuberculosis in Ethiopia is limited. OBJECTIVE: To investigate the molecular characteristics of M. tuberculosis strains implicated in pulmonary TB in the study area. METHODS AND RESULTS: A cross-sectional study was conducted using socio-demographic, clinical and culture data combined with molecular typing analysis. The proportion of TB and M. tuberculosis isolates was not associated with risk factors (P > 0.05). Of 99 sputum samples, 80.8% were culture-positive. Speciation of isolates showed that 88.8% were M. tuberculosis. Further characterisation led to the identification of 27 different spoligotype patterns of M. tuberculosis; the most dominant shared types were SIT149, SIT53 and SIT54. Of the 27 strains, three strains were new and were reported to the SITVIT database. More than two thirds of the strains belonged to the Euro-American lineage. CONCLUSION: This study shows the presence of several clusters and new strains of M. tuberculosis circulating in pulmonary TB patients in the study area, suggesting recent transmission. Nationwide studies are recommended to map the population structure of M. tuberculosis and set control measures.

Caution on the storage of waters and aqueous solutions in plastic containers for hydrogen and oxygen stable isotope analysis

RATIONALE The choice of containers for storage of aqueous samples between their collection, transport and water hydrogen (2H) and oxygen (18O) stable isotope analysis is a topic of concern for a wide range of fields in environmental, geological, biomedical, food, and forensic sciences. The transport and separation of water molecules during water vapor or liquid uptake by sorption or solution and the diffusive transport of water molecules through organic polymer material by permeation or pervaporation may entail an isotopic fractionation. An experiment was conducted to evaluate the extent of such fractionation. METHODS Sixteen bottle-like containers of eleven different organic polymers, including low and high density polyethylene (LDPE and HDPE), polypropylene (PP), polycarbonate (PC), polyethylene terephthalate (PET), and perfluoroalkoxy-Teflon (PFA), of different wall thickness and size were completely filled with the same mineral water and stored for 659?days under the same conditions of temperature and humidity. Particular care was exercised to keep the bottles tightly closed and prevent loss of water vapor through the seals. RESULTS Changes of up to +5 parts per thousand for d2H values and +2.0 parts per thousand for d18O values were measured for water after more than 1?year of storage within a plastic container, with the magnitude of change depending mainly on the type of organic polymer, wall thickness, and container size. The most important variations were measured for the PET and PC bottles. Waters stored in glass bottles with Polyseal (TM) cone-lined PP screw caps and thick-walled HDPE or PFA containers with linerless screw caps having an integrally molded inner sealing ring preserved their original d2H and d18O values. The carbon, hydrogen, and oxygen stable isotope compositions of the organic polymeric materials were also determined. CONCLUSIONS The results of this study clearly show that for precise and accurate measurements of the water stable isotope composition in aqueous solutions, rigorous sampling and storage procedures are needed both for laboratory standards and for unknown samples. Copyright (c) 2012 John Wiley & Sons, Ltd.

Biotic Factors Affecting Expression of the 2,4-Diacetylphloroglucinol Biosynthesis Gene phlA in Pseudomonas fluorescens Biocontrol Strain CHA0 in the Rhizosphere.

ABSTRACT Production of the polyketide antimicrobial metabolite 2,4-diacetyl-phloroglucinol (DAPG) is a key factor in the biocontrol activity of Pseudomonas fluorescens CHA0. Strain CHA0 carrying a translational phlA'-'lacZ fusion was used to monitor expression of the phl biosynthetic genes in vitro and in the rhizosphere. Expression of the reporter gene accurately reflected actual production of DAPG in vitro and in planta as determined by direct extraction of the antimicrobial compound. In a gnotobiotic system containing a clay and sand-based artificial soil, reporter gene expression was significantly greater in the rhizospheres of two monocots (maize and wheat) compared with gene expression in the rhizospheres of two dicots (bean and cucumber). We observed this host genotype effect on bacterial gene expression also at the level of cultivars. Significant differences were found among six additional maize cultivars tested under gnotobiotic conditions. There was no difference between transgenic maize expressing the Bacillus thuringiensis insecticidal gene cry1Ab and the near-isogenic parent line. Plant age had a significant impact on gene expression. Using maize as a model, expression of the phlA'-'lacZ reporter gene peaked at 24 h after planting of pregerminated seedlings, and dropped to a fourth of that value within 48 h, remaining at that level throughout 22 days of plant growth. Root infection by Pythium ultimum stimulated bacterial gene expression on both cucumber and maize, and this was independent of differences in rhizosphere colonization on these host plants. To our knowledge, this is the first comprehensive evaluation of how biotic factors that commonly confront bacterial inoculants in agricultural systems (host genotype, host age, and pathogen infection) modulate the expression of key biocontrol genes for disease suppression.