Anàlisi polifàsica de soques de "Pseudomonas fluorescens" potencials agents de biocontrol de malalties de fruiters

Molts bacteris del grup fluorescent del gènere Pseudomonas són capaços de controlar malalties de les plantes causades per fongs i bacteris fitopatògens (ACBs) o mostren activitat com a bacteris promotors del creixement de les plantes (BPCPs). S'han descrit diversos metabòlits que intervenen de manera important en la seva activitat com a ACBs i BPCPs entre els quals en destaquen el 2,4-diacetilfloroglucinol (Phl), àcid fenazin-1-carboxílic (PCA), Pirrolnitrina (Prn), àcid cianhídric (HCN), àcid 3-indolacètic (IAA), sideròfors i quitinases. L'objectiu principal del nostre treball ha estat la comparació de les característiques d'un grup de Pseudomonas del grup fluorescent utilitzant una aproximació polifàsica amb la finalitat d'establir possibles relacions entre algunes de les característiques i la capacitat d'actuar com a ACB o BPCP. Atesa la importància en el biocontrol de la producció de metabòlits com Phl, PCA i Prn, l'objectiu preliminar ha estat la recerca i obtenció de soques productores d'aquests metabòlits. Per assolir aquest objectiu s'ha emprat una aproximació molecular basada en la detecció dels gens biosintètics implicats en la seva producció en lloc de la detecció directa dels metabòlits per evitar els efectes que poden tenir les condicions de cultiu en la inducció o repressió de la seva síntesi. S'han realitzat diferents protocols basats (i) en la cerca assistida de productors mitjançant l'ús de marcadors fenotípics i posterior confirmació per PCR i, (ii) en l'ús de la PCR per a la detecció dels gens directament dels extractes bacterians, d'enriquiments d'aquests extractes i la realització de la hibridació en colònies per al posterior aïllament. La cerca assistida de productors de Phl mitjançant marcadors fenotípics i posteriorment la utilització de tècniques moleculars (amplificació per PCR del gen phlD), ha estat el millor mètode en el tipus de mostres processades en el nostre treball, on la proporció de productors és relativament baixa. En total s'han aïllat a partir de diversos ambients 4 soques portadores dels gens de la síntesi de PCA, 15 de Phl i 1 de Prn. S'ha constituït una col·lecció de 72 soques de Pseudomonas del grup fluorescent que inclou 18 aïllats propis portadors dels gens biosintètics necessaris per la producció de Phl PCA i Prn; 6 soques de referència procedents de col·leccions de cultius tipus, 14 soques productores dels diferents antibiòtics cedides per altres investigadors i una selecció de 34 soques procedents d'un treball previ realitzat en el nostre grup de recerca. A la col·lecció s'hi troben soques candidates a ACB i BPCP de diverses malalties i plantes. Les 72 soques s'han caracteritzat fenotípica i genotípicament. La caracterització fenotípica s'ha portat a terme mitjançant la identificació a nivell d'espècie amb galeries API 20NE i proves bioquímiques específiques; la producció de metabòlits com PCA, Phl, Prn, IAA, HCN, quitinases i sideròfors mitjançant l'ús de diferents tècniques; antagonisme in vitro en diversos medis enfront dos fongs (Stemphylium vesicarium i Penicillium expansum) i tres bacteris fitopatògens (Erwinia amylovora, Pseudomonas syringae pv. syringae i Xanthomonas arboricola pv. juglandis); l'eficàcia de la inhibició de la infecció en bioassaigs in vivo sobre material vegetal enfront els fongs P. expansum en poma i S. vesicarium en fulles de perera i enfront el bacteri E. amylovora en fruits immadurs de perera i, finalment, en assaigs de promoció de creixement en dos portaempelts comercials de Prunus. Cal destacar que P. expansum causa la podridura blava en pomes i peres en postcollita, S. vesicarium la taca bruna de la perera i E. amylovora el foc bacterià de les rosàcies. El nombre de soques de Pseudomonas, sobre el total de les 72 estudiades, productores d'IAA (4) i quitinases (6) és baix, mentre que és elevat en el cas del HCN (32), que a més està associat a la producció de Phl. Els resultats obtinguts en l'antagonisme in vitro han mostrat en el cas dels bacteris que és dependent del patogen indicador i del medi de cultiu. La presència o absència de ferro no sembla ser un factor que potencií l'antagonisme. En el cas dels fongs no s'ha observat però, influència del medi de cultiu emprat. En el total de 72 soques s'ha observat un percentatge baix de soques que manifesten antagonisme en tots els medis assajats vers 3 o 4 dels patògens (7). Solament 2 d'aquestes 7 soques han mostrat ser també efectives en bioassaigs d'inhibició de les infeccions causades per 2 dels 3 patògens assajats. Algunes de les soques efectives en els bioassaigs no són antagonistes in vitro en cap dels medis assajats enfront el mateix patogen. En el cas de la promoció del creixement, s'han observat més soques promotores del creixement del portaempelts de prunera Marianna 2624 que no en l'híbrid de presseguer-ametller GF677 i les eficàcies assolides són també majors en el cas de Marianna 2624, detectant una elevada especificitat soca/portaempelts La caracterització genotípica s'ha realitzat mitjançant l'anàlisi dels polimorfismes en la longitud dels fragments de restricció de DNA ribosomal (RFLP-rDNA) i l'anàlisi dels polimorfismes en la longitud dels fragments de macrorestricció genòmica de DNA cromosòmic separats per electroforesi en camp polsant (MRFLP-PFGE). Ambdues anàlisis van mostrar una gran heterogeneïtat genètica entre les soques caracteritzades i no s'ha pogut relacionar les agrupacions obtingudes amb les característiques fenotípiques o capacitat d'actuar com a ACB o BPCP. Els patrons de macrorestricció genòmica (MRFLP-PFGE) del bacteri model P. fluorescens EPS288 són estables en el temps i independents de les condicions de cultiu assajades al laboratori o en mostres naturals, mostrant ser una tècnica eficaç en la identificació de reaïllats de mostres naturals inoculades prèviament amb el bacteri. Una selecció de soques que comparteixen el fet de produir floroglucinol s'han caracteritzat mitjançant RFLP i seqüenciació del gen phlD. S'ha establert una relació entre les agrupacions obtingudes en les anàlisis RFLP-rDNA, RFLP-phlD i les seqüències del gen. En l'anàlisi filogenètica de les seqüències del gen phlD s'ha observat un elevat grau de polimorfisme obtenint-se 3 agrupacions principals. Les agrupacions semblen relacionar-se amb els patrons de producció de metabòlits (Phl, HCN i Prn en una primera agrupació; Phl i HCN en la segona i solament Phl en la tercera), però aquestes no s'han pogut relacionar amb l'origen geogràfic de les soques o la seva activitat com a ACBs i/o BPCP. Amb les dades obtingudes de la caracterització fenotípica i genotípica s'ha realitzat una anàlisi multivariant (correspondències, correlacions d'Spearman i de freqüències amb variables categòriques). S'ha demostrat la importància de disposar d'una tècnica que permeti depurar una col·lecció de soques descartant les soques genèticament idèntiques, ja que influeixen en els resultats de les anàlisis. Pels tres patògens assajats com a indicadors i els dos portaempelts emprats, no s'ha observat cap correlació entre la inhibició de la infecció o la promoció del creixement amb les característiques fenotípiques i genotípiques de les soques que fos significatiu i consistent en les tres tècniques emprades.

Pseudomonas fluorescens lipase immobilization on polysiloxane-polyvinyl alcohol composite chemically modified with epichlorohydrin

The technique based on sol-gel approach was used to generate silica matrices derivatives by hydrolysis of silane compounds. The present work evaluates a hybrid matrix obtained with tetraethoxysilane (TEOS) and polyvinyl alcohol (PVA) on the immobilization yield of lipase from Pseudomonas fluorescens. The resulting polysiloxane-polyvinyl alcohol (POS-PVA) matrix combines the property of PVA as a suitable polymer to retain proteins with an excellent optical, thermal and chemical stability of the host silicon oxide matrix. Aiming to render adequate functional groups to the covalent binding with the enzyme the POS-PVA matrix was chemically modified using epichlorohydrin. The results were compared with immobilized derivative on POS-PVA activated with glutaraldehyde. Immobilization yield based on the recovered lipase activity depended on the activating agent and the highest efficiency (32%) was attained when lipase was immobilized on POS-PVA activated with epichlorohydrin, which, probably, provided more linkage points for the covalent bind of the enzyme on the support. This was confirmed by determining the morphological properties using different techniques as X-ray diffraction and scanning electron microscopy (SEM). Comparative studies were carried out to attain optimal activities for free lipase and immobilized systems. For this purpose, a central composite experimental design with different combinations of pH and temperature was performed. Enzymatic hydrolysis with the immobilized enzyme in the framework of the Michaelis-Menten mechanism was also reported. Under optimum conditions, the immobilized derivative on POS-PVA activated with epichlorohydrin showed to have more affinity for the substrate in the hydrolysis of olive oil, with a Michaelis-Menten constant value (K-m) of 293 mM, compared to the value of 401 mM obtained for the immobilized lipase on support activated with glutaraldehyde. Data generated by DSC showed that both immobilized derivatives have similar thermal stabilities. (C) 2007 Elsevier B.V. All rights reserved.

TonB-dependent outer-membrane proteins and siderophore utilization in Pseudomonas fluorescens Pf-5

The soil bacterium Pseudomonas fluorescens Pf-5 produces two siderophores, a pyoverdine and enantio-pyochelin, and its proteome includes 45 TonB-dependent outer-membrane proteins, which commonly function in uptake of siderophores and other substrates from the environment. The 45 proteins share the conserved beta-barrel and plug domains of TonB-dependent proteins but only 18 of them have an N-terminal signaling domain characteristic of TonB-dependent transducers (TBDTs), which participate in cell-surface signaling systems. Phylogenetic analyses of the 18 TBDTs and 27 TonB-dependent receptors (TBDRs), which lack the N-terminal signaling domain, suggest a complex evolutionary history including horizontal transfer among different microbial lineages. Putative functions were assigned to certain TBDRs and TBDTs in clades including well-characterized orthologs from other Pseudomonas spp. A mutant of Pf-5 with deletions in pyoverdine and enantio-pyochelin biosynthesis genes was constructed and characterized for iron-limited growth and utilization of a spectrum of siderophores. The mutant could utilize as iron sources a large number of pyoverdines with diverse structures as well as ferric citrate, heme, and the siderophores ferrichrome, ferrioxamine B, enterobactin, and aerobactin. The diversity and complexity of the TBDTs and TBDRs with roles in iron uptake clearly indicate the importance of iron in the fitness and survival of Pf-5 in the environment.

Surface phenomena and hydrodynamic effects on the deposition of Pseudomonas fluorescens

Biofilm adhesion to metals (copper, aluminium and brass) was studied at two different velocities and pH values of 7 and 9. Both bacteria and metals showed negative surface charges at those values of pH, which tends to slow down adhesion. Film densities increased with the fluid velocity and were also affected by the pH and by the growth rate of the bacteria. Long duration tests based on heat transfer measurements were run at five different fluid velocities and at pH = 7, showing in general an asymptotic behaviour and a control of deposition by adhesion and growth phenomena.

Predator-prey chemical warfare determines the expression of biocontrol genes by rhizosphere-associated Pseudomonas fluorescens.

Soil bacteria are heavily consumed by protozoan predators, and many bacteria have evolved defense strategies such as the production of toxic exometabolites. However, the production of toxins is energetically costly and therefore is likely to be adjusted according to the predation risk to balance the costs and benefits of predator defense. We investigated the response of the biocontrol bacterium Pseudomonas fluorescens CHA0 to a common predator, the free-living amoeba Acanthamoeba castellanii. We monitored the effect of the exposure to predator cues or direct contact with the predators on the expression of the phlA, prnA, hcnA, and pltA genes, which are involved in the synthesis of the toxins, 2,4-diacetylphloroglucinol (DAPG), pyrrolnitrin, hydrogen cyanide, and pyoluteorin, respectively. Predator chemical cues led to 2.2-, 2.0-, and 1.2-fold increases in prnA, phlA, and hcnA expression, respectively, and to a 25% increase in bacterial toxicity. The upregulation of the tested genes was related to the antiprotozoan toxicity of the corresponding toxins. Pyrrolnitrin and DAPG had the highest toxicity, suggesting that bacteria secrete a predator-specific toxin cocktail. The response of the bacteria was elicited by supernatants of amoeba cultures, indicating that water-soluble chemical compounds were responsible for induction of the bacterial defense response. In contrast, direct contact of bacteria with living amoebae reduced the expression of the four bacterial toxin genes by up to 50%, suggesting that protozoa can repress bacterial toxicity. The results indicate that predator-prey interactions are a determinant of toxin production by rhizosphere P. fluorescens and may have an impact on its biocontrol potential.

Oral insecticidal activity of root colonizing Pseudomonas fluorescens CHA0: a biocontrol agent with potential to control plant pests and diseases

The application of microbial biocontrol agents for the control of fungal plant diseases and plant insect pests is a promising approach in the development of environmentally benign pest management strategies. The ideal biocontrol organism would be a bacterium or a fungus with activity against both, insect pests and fungal pathogens. Here we demonstrate the oral insecticidal activity of the root colonizing Pseudomonas fluorescens CHA0, which is so far known for its capacity to efficiently suppress fungal plant pathogens. Feeding assays with CHA0-sprayed leaves showed that this strain displays oral insecticidal activity and is able to efficiently kill larvae of three important insect pests. We further show data indicating that the Fit insect toxin produced by CHA0 and also metabolites controlled by the global regulator GacA contribute to oral insect toxicity.

Amplification of the housekeeping sigma factor in Pseudomonas fluorescens CHA0 enhances antibiotic production and improves biocontrol abilities.

Pseudomonas fluorescens CHA0 produces a variety of secondary metabolites, in particular the antibiotics pyoluteorin and 2,4-diacetylphloroglucinol, and protects various plants from diseases caused by soilborne pathogenic fungi. The rpoD gene encoding the housekeeping sigma factor sigma 70 of P. fluorescens was sequenced. The deduced RpoD protein showed 83% identity with RpoD of Pseudomonas aeruginosa and 67% identity with RpoD of Escherichia coli. Attempts to inactivate the single chromosomal rpoD gene of strain CHA0 were unsuccessful, indicating an essential role of this gene. When rpoD was carried by an IncP vector in strain CHA0, the production of both antibiotics was increased severalfold and, in parallel, protection of cucumber against disease caused by Pythium ultimum was improved, in comparison with strain CHA0.

Role of the stress sigma factor RpoS in GacA/RsmA-controlled secondary metabolism and resistance to oxidative stress in Pseudomonas fluorescens CHA0.

In Pseudomonas fluorescens biocontrol strain CHA0, the two-component system GacS/GacA positively controls the synthesis of extracellular products such as hydrogen cyanide, protease, and 2,4-diacetylphloroglucinol, by upregulating the transcription of small regulatory RNAs which relieve RsmA-mediated translational repression of target genes. The expression of the stress sigma factor sigmaS (RpoS) was controlled positively by GacA and negatively by RsmA. By comparison with the wild-type CHA0, both a gacS and an rpoS null mutant were more sensitive to H2O2 in stationary phase. Overexpression of rpoS or of rsmZ, encoding a small RNA antagonistic to RsmA, restored peroxide resistance to a gacS mutant. By contrast, the rpoS mutant showed a slight increase in the expression of the hcnA (HCN synthase subunit) gene and of the aprA (major exoprotease) gene, whereas overexpression of sigmaS strongly reduced the expression of these genes. These results suggest that in strain CHA0, regulation of exoproduct synthesis does not involve sigmaS as an intermediate in the Gac/Rsm signal transduction pathway whereas sigmaS participates in Gac/Rsm-mediated resistance to oxidative stress.

Biocontrol strain Pseudomonas fluorescens CHA0 and its genetically modified derivative with enhanced biocontrol capability exert comparable effects on the structure of a Sinorhizobium meliloti population in a gnotobiotic system

The impact of biocontrol strain Pseudomonas fluorescens CHA0 and of its genetically modified, antibiotic-overproducing derivative CHA0/pME3424 on a reconstructed population of the plant-beneficial Sinorhizobium meliloti bacteria was assessed in gnotobiotic systems. In sterile soil, the final density of the reconstructed S. meliloti population decreased by more than one order of magnitude in the presence of either of the Pseudomonas strains when compared to a control without addition of P. fluorescens. Moreover, there was a change in the proportion of each individual S. meliloti strain within the population. Plant tests also revealed changes in the nodulating S. meliloti population in the presence of strains CHA0 or CHA0/pME3424. In both treatments one S. meliloti strain, f43, was significantly reduced in its root nodule occupancy. Analysis of alfalfa yields showed a slight but statistically significant increase in shoot dry weight when strain CHA0 was added to the reconstructed S. meliloti population whereas no such effect was observed with CHA0/pME3424.

Oxygen-sensing reporter strain of Pseudomonas fluorescens for monitoring the distribution of low-oxygen habitats in soil.

The root-colonizing bacterium Pseudomonas fluorescens CHA0 was used to construct an oxygen-responsive biosensor. An anaerobically inducible promoter of Pseudomonas aeruginosa, which depends on the FNR (fumarate and nitrate reductase regulation)-like transcriptional regulator ANR (anaerobic regulation of arginine deiminase and nitrate reductase pathways), was fused to the structural lacZ gene of Escherichia coli. By inserting the reporter fusion into the chromosomal attTn7 site of P. fluorescens CHA0 by using a mini-Tn7 transposon, the reporter strain, CHA900, was obtained. Grown in glutamate-yeast extract medium in an oxystat at defined oxygen levels, the biosensor CHA900 responded to a decrease in oxygen concentration from 210 x 10(2) Pa to 2 x 10(2) Pa of O(2) by a nearly 100-fold increase in beta-galactosidase activity. Half-maximal induction of the reporter occurred at about 5 x 10(2) Pa. This dose response closely resembles that found for E. coli promoters which are activated by the FNR protein. In a carbon-free buffer or in bulk soil, the biosensor CHA900 still responded to a decrease in oxygen concentration, although here induction was about 10 times lower and the low oxygen response was gradually lost within 3 days. Introduced into a barley-soil microcosm, the biosensor could report decreasing oxygen concentrations in the rhizosphere for a 6-day period. When the water content in the microcosm was raised from 60% to 85% of field capacity, expression of the reporter gene was elevated about twofold above a basal level after 2 days of incubation, suggesting that a water content of 85% caused mild anoxia. Increased compaction of the soil was shown to have a faster and more dramatic effect on the expression of the oxygen reporter than soil water content alone, indicating that factors other than the water-filled pore space influenced the oxygen status of the soil. These experiments illustrate the utility of the biosensor for detecting low oxygen concentrations in the rhizosphere and other soil habitats.

Regulation of the Gac/Rsm pathway in "Pseudomonas fluorescens" CHA0

SUMMARY : Two-component systems are key mediators implicated in the response of numerous bacteria to a wide range of signals and stimuli. The two-component system comprised of the sensor kinase GacS and the response regulator GacA is broadly distributed among γ-proteobacteria bacteria and fulfils diverse functions such as regulation of carbon storage and expression of virulence. In Pseudomonas fluorescens, a soil bacterium which protects plants from root-pathogenic fungi and nematodes, the GacS/GacA two-component system has been shown to be essential for the production of secondary metabolites and exoenzymes required for the biocontrol activity of the bacterium. The regulatory cascade initiated by GacS/GacA consists of two translational repressor proteins, RsmA and RsmE, as well as three GacAcontrolled small regulatory RNAs RsmX, RsmY and RsmZ, which titrate RsmA and RsmE to allow the expression of biocontrol factors. Genetic analysis revealed that two additional sensor kinases termed RetS and Lads were involved as negative and positive control elements, respectively, in the Gac/Rsm pathway in P. fluoresens CHAO. Furthermore, it could be proposed that RetS and Lads interact with GacS, thereby modulating the expression of antibiotic compounds and hydrogen cyanide, as well as the rpoS gene encoding the stress and stationary phase sigma factor σ. Temperature was found to be an important environmental cue that influences the Gac/Rsm network. Indeed, the production of antibiotic compounds and hydrogen cyanide was reduced at 35°C, by comparison with the production at 30°C. RetS was identified to be involved in this temperature control. The small RNA RsmY was confirmed to be positively regulated by GacA and RsmA/RsmE. Two essential regions were identified in the rsmY promoter by mutational analysis, the upstream activating sequence (UAS) and the linker sequence. Although direct experimental evidence is still missing, several observations suggest that GacA may bind to the UAS, whereas the linker region would be recognized by intermediate RsmA/RsmEdependent repressors and/or activators. In conclusion, this work has revealed new elements contributing to the function of the signal transduction mechanisms in the Gac/Rsm pathway. RESUME : Les systèmes ä deux composants sont des mécanismes d'une importance notoire que beaucoup de bactéries utilisent pour faire face et répondre aux stimuli environnementaux. Le système à deux composants comprenant le senseur GacS et le régulateur de réponse GacA est très répandu chez les γ-protéobactéries et remplit des fonctions aussi diverses que la régulation du stockage de carbone ou l'expression de la virulence. Chez Pseudomonas fluorescens CHAO, une bactérie du sol qui protège les racines des plantes contre des attaques de champignons et nématodes pathogènes, le système à deux composants GacS/GacA est essentiel à la production de métabolites secondaires et d'exoenzymes requis pour l'activité de biocontrôle de la bactérie. La cascade régulatrice initiée pas GacS/GacA fait intervenir deux protéines répresseur de traduction, RsmA et RsmE, ainsi que trois petits ARNs RsmX, RsmY et RsmZ, dont la production est contrôlée par GacA. Ces petits ARNs ont pour rôle de contrecarrer l'action des protéines répressseur de la traduction, ce qui permet l'expression de facteurs de biocontrôle. Des analyses génétiques ont révélé la présence de deux senseurs supplémentaires, appelés Rets et Lads, qui interviennent dans la cascade Gac/Rsm de P. fluorescens. L'impact de ces senseurs est, respectivement, négatif et positif. Ces interactions ont apparenunent lieu au niveau de GacS et permettent une modulation de l'expression des antibiotiques et de l'acide cyanhydrique, ainsi que du gène rpoS codant pour le facteur sigma du stress. La température s'est révélée être un facteur environnemental important qui influence la cascade Gac/Rsm. Il s'avère en effet que la production d'antibiotiques ainsi que d'acide cyanhydrique est moins importante à 35°C qu'à 30°C. L'implication du senseur Rets dans ce contrôle par la température a pu être démontrée. La régulation positive du petit ARN RsmY par GacA et RsmA/RsmE a pu être confirmée; par le biais d'une analyse mutationelle, deux régions essentielles ont pu être mises en évidence dans la région promotrice de rsmY. Malgré le manque de preuves expérimentales directes, certains indices suggèrent que GacA puisse directement se fixer sur une des deux régions (appelée UAS), tandis que la deuxième région (appelée linker) serait plutôt reconnue par des facteurs intermédiaires (activateurs ou répresseurs) dépendant de RsmA/RsmE. En conclusion, ce travail a dévoilé de nouveaux éléments permettant d'éclairer les mécanismes de transduction des signaux dans la cascade Gac/Rsm.

Predominance of Nonculturable Cells of the Biocontrol Strain Pseudomonas fluorescens CHA0 in the Surface Horizon of Large Outdoor Lysimeters.

The persistence of the biocontrol agent Pseudomonas fluorescens CHA0 in the surface horizon of 12 large outdoor lysimeters planted with winter wheat, Phacelia tanacetifolia followed by spring wheat, or maize was monitored for 1 year. Soil was inoculated with a spontaneous rifampin-resistant mutant (CHA0-Rif) of CHA0, and the strain was studied by using colony counts, Kogure's direct viable counts, and total counts (immunofluorescence). The number of culturable cells of the inoculant decreased progressively from 8 to 2 log CFU/g of soil or lower. However, culturable cells of CHA0-Rif accounted for less than 1% of the total cells of the inoculant 8 months after release in autumn. Since viable but nonculturable cells represented less than a quarter of the latter, most cells of CHA0-Rif in soil were thus inactive-dormant or dead at that time. Nonculturable cells of the inoculant were predominant also in the surface horizon of the lysimeters inoculated in the spring, and a significant fraction of them were viable. Results suggest that the occurrence of nonculturable cells of CHA0-Rif was influenced by climatic factors (water availability and soil temperature) and the abundance of roots in soil. The fact that the inoculant persisted as mixed populations of cells of different physiological states, in which nonculturable cells were predominant, needs to be taken into account when assessing the autecology of wild-type or genetically modified pseudomonads released into the soil ecosystem.