Influence of oxygen conditions on bacterial interactions within biofilms related with cystic fibrosis

Disserta����o de mestrado em Bioengineering

Insights into bacterial colony morphology evolution and diversification during infection development in cystic fibrosis

Tese de Doutoramento em Engenharia Biom��dica.

Evaluation of antimicrobial strategies against biomaterial-associated infections: impact of the surrounding environmental conditions

Disserta����o de mestrado em Bioengenharia

Aspectos moleculares del metabolismo de colina en <i>Pseudomona aeruginosa</i>

Con la realizaci��n de este proyecto se pretende contribuir al conocimiento del metabolismo de colina y de otros compuestos de amonio cuaternario en <i> Pseudomonas aeruginosa </i>. Se enfocar�� el estudio mediante el abordaje microbiol��gico y molecular. Se buscar��n, mediante mutag��nesis qu��mica y transposicional, cepas bacterianas alteradas en el metabolismo de colina. Se tendr��n en cuenta las que presenten fenotipos negativos en su capacidad de producir fosfatasa ��cida, de transportar colina y de osmoprotegerse con beta��na. Se caracterizar��n las mutantes obtenidas anteriormente a este proyecto (cepa Pa10 y Pa5, afectadas en actividad beta��na homociste��na metiltransferasa y colinesterasa, respectivamente) y las pr��ximas por obtener en el laboratorio. Se proceder�� al an��lisis molecular mediante el rescate del gen interrumpido por Tn5-751 o Tn5-phoA. Se construir��n sondas espec��ficas para tal fin. Se realizar��n estudios de complementaci��n en <i> E. coli </i> DH5, utilizando vectores adecuados, mediante t��cnicas ampliamentes citadas en la bibliograf��a. Nuestra principal intenci��n es demostrar si existe una relaci��n entre los operones involucrados en la degradaci��n de colina (genes chu), la s��ntesis de ChE, Ac.Pasa y PLC con los regulones involucrados en la disponibilidad de fosfato (Pho) y de nitr��geno (Ntr).

Preservaci��n de la informaci��n g��nica en bacterias; mecanismos y modulaci��n molecular. Preservation of the genetic information in bacteria; mechanisms and molecular modulation.

El objetivo general del presente proyecto es contribuir a la caracterizaci��n gen��tica y bioqu��mica molecular de mecanismos involucrados en el mantenimiento de la informaci��n g��nica, a trav��s del estudio de sistemas fisiol��gicos involucrados en la prevenci��n, reparaci��n y tolerancia de mutaciones. Dichos sistemas se encuentran evolutivamente conservados y ampliamente distribuidos en los seres vivos. La importancia de los mismos se refleja en el hecho que su deficiencia genera en humanos, enfermedades gen��ticas, apoptosis y c��ncer; y en especies procariotas, c��lulas denominadas "hipermutadoras". En los ��ltimos a��os el estudio de la hipermutabilidad en bacterias ha cobrado gran inter��s ya que se le atribuye importancia en procesos infectivos y en aspectos b��sicos relacionados a evoluci��n. Nuestro modelo de estudio son las bacterias Pseudomonas aeruginosa y Escherichia coli, siendo esta ��ltima especie no solo modelo de estudio sino tambi��n especie de referencia. P. aeruginosa es una bacteria ambiental gram negativa, e importante pat��geno oportunista de humanos. Espec��ficamente nos proponemos estudiar en P. aeruginosa algunos aspectos particulares del Sistema de Reparaci��n de Bases Apareadas Incorrectamente (Mismatch Repair System, MRS), del Sistema de Prevenci��n/Reparaci��n de Lesiones Oxidativas generadas a trav��s de 8-oxo-7,8-dihidroguanina (8-oxo-dG �� GO) y el papel de las ADN Polimerasas de baja fidelidad en la modulaci��n de la tasa de mutaci��n. Asimismo estamos interesados en estudiar en cepas de E. coli deficientes en el sistema Dam, la existencia de subpoblaciones de alta estabilidad gen��tica debido a la eliminaci��n de posibles mutantes por incremento de la expresi��n de los otros componentes del MRS. Metodol��gicamente la caracterizaci��n bioqu��mica de factores proteicos se llevar�� a cabo utilizando prote��nas recombinantes purificadas, an��lisis de interacci��n prote��na-prote��na y prote��na-ADN mediante electroforesis en geles y resonancia plasm��nica de superficie (Biacore), mutagen��sis dirigida in vitro, y estudios de complementaci��n en cepas mutantes espec��ficas. Aspectos fenot��picos y de regulaci��n g��nica en cultivos de biofilm y c��lulas en suspensi��n ser��n estudiados mediante la construcci��n de cepas mutantes, fusiones transcripcionales, PCR en tiempo real, western blot y microscopia de fluorescencia confocal.

Producci��n de factores de virulencia por fitopat��genos bacterianos de soja. Utilizaci��n de productos naturales como biocontrol. Production of virulence factors by soybean bacterial phytopathogens. The use of natural products as biocontrolers.

En Argentina el cultivo de soja ocupa el primer lugar en superficie sembrada. El 90% de la producci��n se obtiene en la zona central del pais. La siembra directa favorece la multiplicaci��n y supervivencia de fitopat��genos causantes de tiz��n y p��stula bacterianos. El tiz��n es producido por Pseudomonas syringae pv. glycinea observ��ndose manchas marrones en las hojas. Produce gran variedad de toxinas: coronatina, faseolotoxina, siringomicina, tabtoxina, prote��nas ���nucleation ice���, entre otras, las cuales contribuyen a la clorosis y necrosis. En la infecci��n, adem��s, est��n involucrados exopolisac��ridos (levano y alginato). La celulosa ha sido relacionada en la adhesi��n bacteriana y en la formaci��n de biofilm. La p��stula es causada por Xanthomonas axonopodis pv. glycines. Produce manchas peque��as con una peque��a p��stula de color claro. Libera enzimas como ��-amilasa, proteasa, endo ��-mannanasa, actividad peptol��tica, que degradan componentes vegetales. Xantan, producido por X. axonopodis es uno de los componentes necesarios para la formaci��n de biofilm. Este ��ltimo es considerado un importante factor de virulencia porque proporciona una estrategia de colonizaci��n que otorga mayor resistencia a ambientes desfavorables, tolerancia a antimicrobianos, producci��n de metabolitos y exoenzimas, etc. Actualmente el control de bacterias fitopat��genas se realiza mediante pesticidas con alta toxicidad para los consumidores y el ambiente. Para evitar las bacteriosis en la pr��ctica se sugiere la rotaci��n de cultivos y utilizar semillas certificadas. Se est��n probando compuestos naturales derivados de plantas medicinales como pesticidas; estos se pueden dividir en varias categor��as fitoqu��micas. Varios estudios confirman la actividad antibacteriana, antif��ngica y antiviral de estos productos. Extractos vegetales con alto contenido de flavonoides y aceite esenciales poseen una importante actividad antibacteriana. Adem��s, algunos aceites esenciales podr��an estar incidiendo en la liberaci��n y/o producci��n de biofilm, exopolisac��ridos y exoprote��nas. La gran incidencia de las infecciones por fitopat��genos y las p��rdidas econ��micas que estas acarrean hacen que su control presente grandes dificultades para la agricultura sustentable en soja de nuestro pa��s. En este trabajo se propone estudiar los diferentes factores de virulencia de cepas bacterianas fitopat��genas y evaluar el rol que cumplen en el proceso de la enfermedad en cultivos de soja y desarrollar estrategias para el control de bacteriosis vegetales aplicando productos naturales aislados de plantas arom��ticas. La correcta utilizaci��n de productos antimicrobianos de origen natural aplicados sobre el cultivo y/o sobre las semillas evitar��a la dispersi��n de la enfermedad y la eliminaci��n al medio ambiente de productos contaminantes no deseados. In Argentina, soybean cultivation occupies the first place; 90% of this cereal is produced in the central region of the country. Intensive tillage practices favour multiplication and survival of bacterial phytopathogens causing blight and pustule diseases. Pseudomonas syringae pv. glycinea produce several toxins like coronatine, faseolotoxine, siringomicine, tabtoxine and proteins of nucleation ice that contribute to the develop of chlorosis and necrosis, characteristic of bacterial blight. It also produces levan and alginate, cellulose and biofilm. Pustule disease is caused by Xanthomonas axonopodis pv glycines, which produce enzymes like ��-amilase, protease, endo ��-mannanase, peptolitic activity, xanthan and biofilm. Nowadays the control of phytopathogenic bacteria consists in the application of pesticides that are toxic for the environment and man. Natural products from medicinal plants are a new alternative for the treatment of phytopathogens. Researches made with phytochemical compounds (flavonoids, phenols, quinones, cummarines, essential oils, terpenes) support the antimicrobial activity of these natural products. What is more, these substances could suppress the biofilm, exoproteins and exopolisaccharides formation and release of them. The infections caused by phytopathogens provoke economical loses and its control presents big difficulties in our country. The proposal of this work is the characterization of phytopatoghenic strains, its virulence factors and the role they play in the disease process. The development of a new alternative for the control of vegetable bacteriosis using natural products obtained from aromatic plants and the correct application of them on sown fields or on seeds is also an objective in this work.

Bioremediation of diesel contaminated soils : an investigation into the effects of biosurfactant and organic matter amendments

The aim of this study was to investigate the effects of biosurfactants and organic matter amendments on the bioremediation of diesel contaminated soil. Two strains of Pseudomonas aeruginosa with the ability to produce biosurfactant were isolated from a water and soil sample in Co. Sligo. The first strain, Isolate A, produced a biosurfactant which contained four rhamnose containing compounds, when grown in proteose peptone glucose ammonium salts medium with glucose as the carbon source. Two of the components were identified as rhamnolipid 1 and 2 whilst the other two components were unidentified. The second strain, Isolate GO, when grown in similar conditions produced a biosurfactant which contained only rhamnolipid 2. The type of aeration system used had a significant effect on the abiotic removal of diesel from soil. Forced aeration at a rate of 120L 02/kg soil/ hour resulted in the greatest removal. Over a 112 day incubation period this type o f aeration resulted in the removal o f 48% o f total hexane extractable material. In relation to bioremediation of the diesel contaminated sandy soil, amending the soil with two inorganic nutrients, KH2PO4 and N��4N03, significantly enhanced the removal of diesel, especially the ��- alkanes, when compared to an unamended control. The biosurfactant from Isolate A and a biosurfactant produced by Pseudomonas aeruginosa NCIMB 8628 (a known biosurfactant producer), when applied at a concentration of three times their critical micelle concentration, had a neutral effect on the biod��gradation o f diesel contaminated sandy soil, even in the presence o f inorganic nutrients. It was deduced that the main reason for this neutral effect was because they were both readily biodegraded by the indigenous microorganisms. The most significant removal of diesel occurred when the soils were amended with two organic materials plus the inorganic nutrients. Amendment of the diesel contaminated soil with spent brewery grain (SBG) removed significantly more diesel than amendment with dried molassed sugar beet pulp (DMSBP). After a 108 day incubation period, amendment of the diesel contaminated soil with DMSBP plus inorganic nutrients and SBG plus inorganic nutrients resulted in 72 and 89% removal of diesel range organics (DRO), in comparison to 41% removal of DRO in an inorganic nutrient amended control. The first order kinetic model described the degradation of the different diesel components with high correlation and was used to calculate Vi lives. The V2 life, of the total ��-alkanes in the diesel was reduced from 40 days in the control to 8.5 and 5.1 days in the presence of DMSBP and SBG, respectively. The V2 life o f the unresolved complex mixture (UCM) in the diesel contaminated soil was also significantly reduced in the presence o f the two organics. DMSBP and SBG addition reduced UCM V2 life to 86 and 43 days, respectively, compared to 153 days in the control. The component of diesel whose removal was enhanced the greatest through the organic material amendments was the isoprenoid, pristane, a compound which until recently was thought to be nonbiodegradable and was used as an inert biomarker in oil degradation studies. The V2 life of pristane was reduced from 533 days in the nutrient amended control to 49.5 and 19.5 days in DMSBP and SBG amended soils. These results indicate that the addition o f the DMSBP and SBG to diesel contaminated soil stimulated diesel biod��gradation, probably by enhancing the indigenous diesel degrading microbial population to degrade diesel hydrocarbons, whilst the addition o f biosurfactants had no enhanced effect on the bioremediation process.

S��bre u'a modifica����o do meio de Monteverde

It is well known that the culture media used in the presumptive diagnosis of suspiciuous colonies from plates inoculated with stools for isolation of enteric organisms do not always correctly indicate the major groups of enterobacteria. In an effort to obtain a medium affording more exact indications, several media (1-9) have been tested. Modifications of some of these media have also been tested with the result that a satisfactory modification of Monteverde's medium was finaly selected. This proved to be most satisfactory, affording, as a result of only one inoculation, a complete series of basic indications. The modification involves changes in the formula, in the method of preparation and in the manner of storage. The formulae are: A. Thymol blue indicator: NaOH 0.1/N .............. 34.4 ml; Thymol blue .............. 1.6 g; Water .................... 65.6 ml. B. Andrade's indicator. C. Urea and sugar solution: Urea ..................... 20 g; Lactose ................... 30 g; Sucrose ................... 30 g; Water .................... 100 ml. The mixture (C.) should be warmed slightly in order to dissolve the ingredients rapidly. Sterilise by filtration (Seitz). Keep stock in refrigeratior. The modification of Monteverde's medium is prepared in two parts. Semi-solid part - Peptone (Difco) 2.0 g; NaCl 0.5 g; Agar 0.5 g; Water 100.0 ml. Boil to dissolve the ingredients. Adjust pH with NaOH to 7.3-7.4. Boil again for precipitation. Filter through cotton. Ad indicators "A" 0.3 ml and "B" 1.0 ml. Sterilise in autoclave 115��C, 15 minutes in amounts not higher than 200 ml. Just before using, add solution "C" asseptically in amounts of 10 ml to 200 ml of the melted semi-solid medium, maintained at 48-50��C. Solid part - Peptone (Difco) 1.5 g; Trypticase (BBL) 0.5 g; Agar 2.0 g; Water 100,00 ml. Boil to dissolve the ingredients. Adjust pH with NaOH to 7.3-7.4. Boils again. Filter through cotton. Add indicators "A" 0.3 ml and "B" 1.0 ml; ferrous ammonium sulfate 0.02 g; sodiun thiosulfate 0.02 g. Sterilise in autoclave 115��C, 15 minutes in amounts not higher than 200 ml. Just before using, add solution "C" asseptically in amounts of 10 ml to 200 ml of the melted solid medium, maintained at 48-50��C. Final medium - The semi-solid part is dispensed first (tubes about 12 x 120 mm) in 2.5 ml amounts and left to harden at room temperature, in vertical position. The solid part is dispensed over the hardened semi-solid one in amounts from 2.0 ml to 2.5 ml and left to harden in slant position, affording a butt of 12 to 15 mm. The tubes of medium should be subjected to a sterility test in the incubator, overnight. Tubes showing spontaneous gas bubbles (air) should then be discarded. The medium should be stored in the incubator (37��C), for not more than 2 to 4 days. Storage of the tubes in the ice-box produces the absorption of air which is released as bubbles when the tubes are incubated at 37��C after inoculation. This fact confirmed the observation of ARCHAMBAULT &amp; McCRADY (10) who worked with liquid media and the aplication of their observation was found to be essential to the proper working conditions of this double-layer medium. Inoculation - The inoculation is made by means of a long straight needle, as is usually done on the triple sugar, but the needel should penetrate only to about half of the height of the semi-solid column. Indol detection - After inoculation, a strip of sterelized filter papaer previously moistened with Ehrlich's reagent, is suspended above the surface of the medium, being held between the cotton plug and the tube. Indications given - In addition to providing a mass of organisms on the slant for serological invetigations, the medium gives the following indications: 1. Acid from lactose and/or sucrose (red, of yellowsh with strains which reduce the indicators). 2. Gas from lactose and/or sucrose (bubbles). 3. H[2]S production, observed on the solid part (black). 4. Motility observed on the semi-solid part (tubidity). 5. Urease production, observed on solid and semi-solid parts (blue). 6. Indol production, observed on the strip of filter paper (red or purplish). Indol production is not observed with indol positive strains which rapidly acidify the surface o the slant, and the use of oxalic acid has proved to give less sensitive reaction (11). Reading of results - In most cases overnight incubation is enough; sometimes the reactions appear within only a few hours of incubation, affording a definitive orientation of the diagnosis. With some cultures it is necessary to observe the medium during 48 hours of incubation. A description showing typical differential reaction follows: Salmonella: Color of the medium unchanged, with blackening of the solid part when H[2]S is positive. The slant tends to alkalinity (greenish of bluish). Gas always absent. Indol negative. Motility positive or negative. Shigella: Color of the medium unchanged at the beginning of incubation period, but acquiring a red color when the strain is late lactose/sucrose positive. Slant tending to alkalinity (greenish or purplish). Indol positive or negative. Motility, gas and H[2]S always negative. Proteus: Color of the medium generally changes entirely to blue or sometimes to green (urease positive delayed), with blackening of solid part when H[2]S is positive. Motility positive of negative. Indol positive. Gas positive or negative. The strains which attack rapidly sucrose may give a yellow-greenish color to the medium. Sometimes the intense blue color of the medium renders difficult the reading of the H[2]S production. Escherichiae and Klebsiellae: Color of the medium red or yellow (acid) with great and rapid production of gas. Motility positive or negative. Indol generally impossible to observe. Paracoli: Those lactose of sucrose positive give the same reaction as Esherichia. Those lactose or sucrose negatives give the same reactions as Salmonellae. Sometimes indol positive and H[2]S negative. Pseudomonas: Color of the medium unchanged. The slant tends to alkalinity. It is impossible to observe motility because there is no growth in the bottom. Alkaligenes: Color of the medium unchanged. The slant tends to alkalinity. The medium does not alter the antigenic properties of the strains and with the mass of organisms on the slant we can make the serologic diagnosis. It is admitted that this medium is somewhat more laborious to prepare than others used for similar purposes. Nevertheless it can give informations generally obtained by two or three other media. Its use represents much saving in time, labor and material, and we suggest it for routine laboratory work in which a quick presumptive preliminary grouping of enteric organisms is needed.

The interaction of Gram negative bacteria and S. mansoni in mice with experimental Schistosomiasis

Animals (122 mice) were infected each with eighty cercariae of S. mansoni and subsequently challenged intravenously eight weeks later with the following gram-negative organisms. S. typhi, E. coli, Klebsiella-enterobacter species, Proteus mirabilis and Pseudomonas aeruginosa. Enumeration of bacteria in the liver, spleen and blood and S. mansoni from the portal sistem was performed from one to four weeks later in infected animals. A significant difference between infection produced by S. typhi and other gram negative organisms was observed: S. typhi persisted longer in the spleen and liver and could be recovered from S. mansoni worms up to three weeks following bacterial infection. Other gram negative bacteria disappeared from S. mansoni worms after two weeks of initial challenge. Additional animals (51 mice) infected with S. mansoni were given S. typhi, E. coli or sterile saline. After two weeks, animals were sacrificed and the recovery rate of worms from the portal system, and the mesenteric and hepatic oogram were determined. in animals infected with E. coli a significant decrease in the number of worms was observed compared to the saline control group; thirty worms were recovered in the control group compared to two worms in e. coli infected animals. In addition, the patterns of oviposition was significantly different in these latter animals suggesting complete inhibition of this process. Following S. typhi infection the difference in recovery of worms and pattern of oviposition was minimal. These findings suggest a difference in the interaction of various gram negative bacteria and S. mansoni and are consistent with the clinical observation of prolonged salmonella bacteremia in patients with schistosomiasis.

Bact��rias gram negativas resistentes a antimicrobianos em alimentos

A partir de 154 esp��cimens de alimentos, representados por hortali��as (alface), leite e merenda escolar, obteve-se o isolamento e identifica����o de 400 amostras de bacilos Gram negativos. Esta amostragem se distribuiu em 339 enterobact��rias (Escherichia, Shigella, Citrobacter, Klebsiella, Enterobacter, Serratia e Proteus) e 61 de g��neros afins (Acinetobacter, Flavobacterium, Aeromonas e Pseudomonas). Submetendo-se as culturas aos antimicrobianos: sulfadiazina (Su), estreptomicina (Sm), tetraciclina (Tc), cloranfenicol (Cm), canamicina (Km), ampicilina (Ap), ��cido nalid��xico (Nal) e gentamicina (Gm), observou-se apenas seis estirpes sens��veis a todas as drogas e sensibilidade absoluta �� Gm. A predomin��ncia dos modelos Su (27,6%) e Su-Ap (39,6%) incidiu nas enterobact��rias, enquanto que, 18,0% para Ap e 9,8% para Su-Ap foram detectados nos g��neros afins. Para caracteriza����o da resist��ncia foram realizados testes de conjuga����o e a totalidade das culturas n��o revelou transfer��ncia para o gene que confere resist��ncia ao ��cido nalid��xico. Relevantes s��o as taxas de amostras R+ observadas nos bacilos ent��ricos, oscilando em torno de 90% (leite e merenda escolar) e alface, em torno de 70%

Fitness correlates with the extent of cheating in a bacterium.

There is growing awareness of the importance of cooperative behaviours in microbial communities. Empirical support for this insight comes from experiments using mutant strains, termed 'cheats', which exploit the cooperative behaviour of wild-type strains. However, little detailed work has gone into characterising the competitive dynamics of cooperative and cheating strains. We test three specific predictions about the fitness consequences of cheating to different extents by examining the production of the iron-scavenging siderophore molecule, pyoverdin, in the bacterium Pseudomonas aeruginosa. We create a collection of mutants that differ in the amount of pyoverdin that they produce (from 1% to 96% of the production of paired wild types) and demonstrate that these production levels correlate with both gene activity and the ability to bind iron. Across these mutants, we found that (1) when grown in a mixed culture with a cooperative wild-type strain, the relative fitness of a mutant is negatively correlated with the amount of pyoverdin that it produces; (2) the absolute and relative fitness of the wild-type strain in the mixed culture is positively correlated with the amount of pyoverdin that the mutant produces; and (3) when grown in a monoculture, the absolute fitness of the mutant is positively correlated with the amount of pyoverdin that it produces. Overall, we demonstrate that cooperative pyoverdin production is exploitable and illustrate how variation in a social behaviour determines fitness differently, depending on the social environment.

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.

Synthesis of medium-chain-length polyhydroxyalkanoates in arabidopsis thaliana using intermediates of peroxisomal fatty acid beta-oxidation.

Polyhydroxyalkanoate (PHA) is a family of polymers composed primarily of R-3-hydroxyalkanoic acids. These polymers have properties of biodegradable thermoplastics and elastomers. Medium-chain-length PHAs (MCL-PHAs) are synthesized in bacteria by using intermediates of the beta-oxidation of alkanoic acids. To assess the feasibility of producing MCL-PHAs in plants, Arabidopsis thaliana was transformed with the PhaC1 synthase from Pseudomonas aeruginosa modified for peroxisome targeting by addition of the carboxyl 34 amino acids from the Brassica napus isocitrate lyase. Immunocytochemistry demonstrated that the modified PHA synthase was appropriately targeted to leaf-type peroxisomes in light-grown plants and glyoxysomes in dark-grown plants. Plants expressing the PHA synthase accumulated electron-lucent inclusions in the glyoxysomes and leaf-type peroxisomes, as well as in the vacuole. These inclusions were similar to bacterial PHA inclusions. Analysis of plant extracts by GC and mass spectrometry demonstrated the presence of MCL-PHA in transgenic plants to approximately 4 mg per g of dry weight. The plant PHA contained saturated and unsaturated 3-hydroxyalkanoic acids ranging from six to 16 carbons with 41% of the monomers being 3-hydroxyoctanoic acid and 3-hydroxyoctenoic acid. These results indicate that the beta-oxidation of plant fatty acids can generate a broad range of R-3-hydroxyacyl-CoA intermediates that can be used to synthesize MCL-PHAs.

A New Large-DNA-Fragment Delivery System Based on Integrase Activity from an Integrative and Conjugative Element.

During the past few decades, numerous plasmid vectors have been developed for cloning, gene expression analysis, and genetic engineering. Cloning procedures typically rely on PCR amplification, DNA fragment restriction digestion, recovery, and ligation, but increasingly, procedures are being developed to assemble large synthetic DNAs. In this study, we developed a new gene delivery system using the integrase activity of an integrative and conjugative element (ICE). The advantage of the integrase-based delivery is that it can stably introduce a large DNA fragment (at least 75 kb) into one or more specific sites (the gene for glycine-accepting tRNA) on a target chromosome. Integrase recombination activity in Escherichia coli is kept low by using a synthetic hybrid promoter, which, however, is unleashed in the final target host, forcing the integration of the construct. Upon integration, the system is again silenced. Two variants with different genetic features were produced, one in the form of a cloning vector in E. coli and the other as a mini-transposable element by which large DNA constructs assembled in E. coli can be tagged with the integrase gene. We confirmed that the system could successfully introduce cosmid and bacterial artificial chromosome (BAC) DNAs from E. coli into the chromosome of Pseudomonas putida in a site-specific manner. The integrase delivery system works in concert with existing vector systems and could thus be a powerful tool for synthetic constructions of new metabolic pathways in a variety of host bacteria.

Differential peptide binding to CD40 evokes counteractive responses.

The antigen-presenting cell-expressed CD40 is implied in the regulation of counteractive immune responses such as induction of pro-inflammatory and anti-inflammatory cytokines interleukin (IL)-12 and IL-10, respectively. The mechanism of this duality in CD40 function remains unknown. Here, we investigated whether such duality depends on ligand binding. Based on CD40 binding, we identifed two dodecameric peptides, peptide-7 and peptide-19, from the phage peptide library. Peptide-7 induces IL-10 and increases Leishmania donovani infection in macrophages, whereas peptide-19 induces IL-12 and reduces L. donovani infection. CD40-peptide interaction analyses by surface plasmon resonance and atomic force microscopy suggest that the functional differences are not associated with the studied interaction parameters. The molecular dynamic simulation of the CD40-peptides interaction suggests that these two peptides bind to two different places on CD40. Thus, we suggest for the first time that differential binding of the ligands imparts functional duality to CD40.