A new generation of vectors with increased induction ratios by overimposing a second regulatory level by attenuation

A major drawback of regulated gene expression from vectors bearing strong promoters is the associated high basal expression level. Simple regulatory systems have an intrinsic limitation in the range of induction, and attempts to mutate promoters to reduce basal expression usually result in concomitant reduction of induced levels. We have explored the possibility of reducing basal levels of gene expression while keeping induced levels intact by incorporating an additional regulatory circuit controlling a different step of the expression process. We have integrated the nasFEDCBA transcriptional attenuation system of Klebsiella oxytoca into a cascade expression circuit based on different regulatory elements of Pseudomonas putida, and also into a system based on the tac promoter, to expand their regulatory capacity. Basal expression from the promoters of these circuits was reduced by more than 10-fold by the nasF attenuator sequence while keeping the induced levels intact in the presence of the antiterminator protein, thus increasing the induction ratio by up to 1700-fold. In addition, using different combinations of regulatory elements and inducing conditions, we were able to obtain a broad range of expression levels. These vectors and the concept of their design will be very useful in regulating overproduction of heterologous proteins both at laboratory and industrial scales

Búsqueda de bacterias promotoras del crecimiento vegetal (PGPRs) y evaluación de sus potencialidades en condiciones de cultivo

El modelo de una agricultura más respetuosa con el medio ambiente, en el marco de un desarrollo sostenible, está presentando gran auge, corrigiendo así los desequilibrios de un sistema más productivista, el cual se ha estado llevando a cabo en las últimas décadas. Este tipo de agricultura fomenta un modelo sostenible con un menor deterioro del medio ambiente, mediante la disminución en el uso de agroquímicos. En este contexto, la utilización de microorganismos en la producción de cultivos ha mostrado gran interés, concretamente los biofertilizantes son productos de microorganismos aplicados al suelo o la planta con el fin de sustituir parcial o totalmente la fertilización química, evitando los inconvenientes derivados del uso excesivo de fertilizantes químicos (Armenta-Bojorquez, 2009). Los microorganismos denominados PGPR (del inglés Plant Growth-Promoting Rhizobacteria) son bacterias o hongos que habitan en las raíces de las plantas o muy cerca de ellas e influyen, mediante diferentes mecanismos, de manera positiva en el crecimiento de la planta (Spaepen et al., 2009). El estudio de las potencialidades de diferentes bacterias como PGPR ha sido evaluado por diferentes métodos, como la determinación de la producción de sideróforos, intrínsecamente relacionado con su capacidad para ser utilizada como agentes de biocontrol (Welbaum et al., 2004), cuantificación de AIA (fitohormona encargada de promover el crecimiento vegetal) y determinación de diferentes actividades bacterianas (fosfatasa, celulolasa,…). La evaluación de la eficacia de este tipo de microorganismos en plantas ha sido estudiada, con resultados satisfactorios en algunos casos y con perspectivas de ser utilizados en campo, sustituyendo a productos químicos más nocivos, utilizados en la actualidad

Translational and structural requirements of the early nodulin gene enod40, a short-open reading frame-containing RNA, for elicitation of a cell-specific growth response in the alfalfa root cortex

A diversity of mRNAs containing only short open reading frames (sORF-RNAs; encoding less than 30 amino acids) have been shown to be induced in growth and differentiation processes. The early nodulin gene enod40, coding for a 0.7-kb sORF-RNA, is expressed in the nodule primordium developing in the root cortex of leguminous plants after infection by symbiotic bacteria. Ballistic microtargeting of this gene into Medicago roots induced division of cortical cells. Translation of two sORFs (I and II, 13 and 27 amino acids, respectively) present in the conserved 5* and 3* regions of enod40 was required for this biological activity. These sORFs may be translated in roots via a reinitiation mechanism. In vitro translation products starting from the ATG of sORF I were detectable by mutating enod40 to yield peptides larger than 38 amino acids. Deletion of a Medicago truncatula enod40 region between the sORFs, spanning a predicted RNA structure, did not affect their translation but resulted in significantly decreased biological activity. Our data reveal a complex regulation of enod40 action, pointing to a role of sORF-encoded peptides and structured RNA signals in developmental processes involving sORF-RNA

Differential roles of HypC and HupF proteins for hydrogenase synthesis in Rhizobium leguminosarum.

Some diazotrophic bacteria induce [NiFe] hydrogenases to recycle the hydrogen evolved by nitrogenase during the nitrogen fixation process. Biosynthesis of Rhizobium leguminosarum [FeNi] hydrogenase requires a number of accessory proteins (products of hup and hyp genes) that mediate the incorporation of Ni and Fe into the active site. Among them, HypC-paralog HupF and HupK are present in bacteria that synthesize hydrogenase in the presence of oxygen. Hydrogenase activity in mutant strains lacking either hupF or hypC genes was severely reduced, indicating that both proteins are essentials for biosynthesis of hydrogenase. Co-purification of StrepTag labelled variants of HupF and HypC by an affinity chromatography-based approach demonstrated interactions between HupL-HupF and HypC-HupK. Experiments carried out with strains induced for hydrogenase under 3% oxygen tensions indicated that HupF might provide additional stability to HupL under these condition

Dual role of HupF in the biosynthesis of [NiFe] hydrogenase in Rhizobium leguminosarum

Background: [NiFe] hydrogenases are enzymes that catalyze the oxidation of hydrogen into protons and electrons, to use H2 as energy source, or the production of hydrogen through proton reduction, as an escape valve for the excess of reduction equivalents in anaerobic metabolism. Biosynthesis of [NiFe] hydrogenases is a complex process that occurs in the cytoplasm, where a number of auxiliary proteins are required to synthesize and insert the metal cofactors into the enzyme structural units. The endosymbiotic bacterium Rhizobium leguminosarum requires the products of eighteen genes (hupSLCDEFGHIJKhypABFCDEX) to synthesize an active hydrogenase. hupF and hupK genes are found only in hydrogenase clusters from bacteria expressing hydrogenase in the presence of oxygen. Results: HupF is a HypC paralogue with a similar predicted structure, except for the C-terminal domain present only in HupF. Deletion of hupF results in the inability to process the hydrogenase large subunit HupL, and also in reduced stability of this subunit when cells are exposed to high oxygen tensions. A ?hupF mutant was fully complemented for hydrogenase activity by a C-terminal deletion derivative under symbiotic, ultra low-oxygen tensions, but only partial complementation was observed in free living cells under higher oxygen tensions (1% or 3%). Co-purification experiments using StrepTag-labelled HupF derivatives and mass spectrometry analysis indicate the existence of a major complex involving HupL and HupF, and a less abundant HupF-HupK complex. Conclusions: The results indicate that HupF has a dual role during hydrogenase biosynthesis: it is required for hydrogenase large subunit processing and it also acts as a chaperone to stabilize HupL when hydrogenase is synthesized in the presence of oxygen

Functional roles of HypC and HupK in the biosynthesis of [NiFe] hydrogenase in Rhizobium leguminosarum.

Some rhizobia induce a hydrogen (H2)-uptake system with a [NiFe] hydrogenase along with nitrogenase to recover part of the energy lost as H2. Biosynthesis of NiFe hydrogenases is a process that ocurrs in the cytoplasm, where a number of auxiliary proteins (products of hup and hyp genes) are required to synthesize and insert the metal cofactors into the enzyme structural units. Although HypC is expressed in all hydrogenase systems, HupF and HupK are found only in bacteria that express the hydrogenase in the presence of oxygen (O2). Co-purification experiments have demonstrated HypC-HupK and HypC-HupL interactions. Results have shown that some conserved residues from HypC and HupK play a protective role of hydrogenase against the presence of O2

Effect of the presence of the plant growth promoting rhizobacterium (PGPR) Chryseobacterium balustinum Aur9 and salt stress in the pattern of flavonoids exuded by soybean roots

In this work we studied how biotic and abiotic stresses can alter the pattern of flavonoids exuded by Osumi soybean roots. A routine method was developed for the detection and characterization of the flavonoids present in soybean root exudates using HPLC-MS/MS. Then, a systematic screening of the flavonoids exuded under biotic stress, the presence of a plant growth promoting rhizobacterium, and salt stress was carried out. Results obtained indicate that the presence of Chryseobacterium balustinum Aur9 or 50 mM NaCl changes qualitatively the pattern of flavonoids exuded when compared to control conditions. Thus, in the presence of C. balustinum Aur9, soybean roots did not exude quercetin and naringenin and, under salt stress, flavonoids daidzein and naringenin could not be detected. Soybean root exudates obtained under saline conditions showed a diminished capacity to induce the expression of the nodA gene in comparison to the exudates obtained in the absence of salt. Moreover, lipochitooligosaccharides (LCOs) were not detected or weakly detected when Sinorhizobium fredii SMH12 was grown in the exudates obtained under salt stress conditions or under salt stress in the presence of C. balustinum Au9, respectively.Fil: Dardanelli, Marta Susana. Universidad de Sevilla. Facultad de Farmacia; España. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Fisicoquímicas y Naturales. Departamento de Biología Molecular. Sección Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Manyani, Hamid. Universidad de Sevilla. Facultad de Farmacia; EspañaFil: González Barroso, Sergio. Universidad de Sevilla. Facultad de Farmacia; EspañaFil: Rodríguez Carvajal, Miguel A.. Universidad de Sevilla. Facultad de Farmacia; EspañaFil: Gil Serrano, Antonio M.. Universidad de Sevilla. Facultad de Farmacia; EspañaFil: Espuny, Maria R.. Universidad de Sevilla. Facultad de Farmacia; EspañaFil: López Baena, Francisco Javier. Universidad de Sevilla. Facultad de Farmacia; EspañaFil: Bellogín, Ramon A.. Universidad de Sevilla. Facultad de Farmacia; EspañaFil: Megías, Manuel. Universidad de Sevilla. Facultad de Farmacia; EspañaFil: Ollero, Francisco J.. Universidad de Sevilla. Facultad de Farmacia; Españ

The nodulation of alfalfa by the acid-tolerant Rhizobium sp. strain LPU83 does not require sulfated forms of lipochitooligosaccharide nodulation signals

The induction of root nodules by the majority of rhizobia has a strict requirement for the secretion of symbiosis-specific lipochitooligosaccharides (nodulation factors [NFs]). The nature of the chemical substitution on the NFs depends on the particular rhizobium and contributes to the host specificity imparted by the NFs. We present here a description of the genetic organization of the nod gene cluster and the characterization of the chemical structure of the NFs associated with the broad-host-range Rhizobium sp. strain LPU83, a bacterium capable of nodulating at least alfalfa, bean, and Leucena leucocephala. The nod gene cluster was located on the plasmid pLPU83b. The organization of the cluster showed synteny with those of the alfalfanodulating rhizobia, Sinorhizobium meliloti and Sinorhizobium medicae. Interestingly, the strongest sequence similarity observed was between the partial nod sequences of Rhizobium mongolense USDA 1844 and the corresponding LPU83 nod genes sequences. The phylogenetic analysis of the intergenic region nodEG positions strain LPU83 and the type strain R. mongolense 1844 in the same branch, which indicates that Rhizobium sp. strain LPU83 might represent an early alfalfa-nodulating genotype. The NF chemical structures obtained for the wild-type strain consist of a trimeric, tetrameric, and pentameric chitin backbone that shares some substitutions with both alfalfa- and bean-nodulating rhizobia. Remarkably, while in strain LPU83 most of the NFs were sulfated in their reducing terminal residue, none of the NFs isolated from the nodH mutant LPU83-H were sulfated. The evidence obtained supports the notion that the sulfate decoration of NFs in LPU83 is not necessary for alfalfa nodulation.Instituto de Biotecnologia y Biologia Molecula

Toward the Assessment of Food Toxicity for Celiac Patients: Characterization of Monoclonal Antibodies to a Main Immunogenic Gluten Peptide

13 pages, 8 figures.-- PMID: 18509534 [PubMed].-- PMCID: PMC2386552.[Background and Aims] Celiac disease is a permanent intolerance to gluten prolamins from wheat, barley, rye and, in some patients, oats. Partially digested gluten peptides produced in the digestive tract cause inflammation of the small intestine. High throughput, immune-based assays using monoclonal antibodies specific for these immunotoxic peptides would facilitate their detection in food and enable monitoring of their enzymatic detoxification. Two monoclonal antibodies, G12 and A1, were developed against a highly immunotoxic 33-mer peptide. The potential of each antibody for quantifying food toxicity for celiac patients was studied.[Methods] Epitope preferences of G12 and A1 antibodies were determined by ELISA with gluten-derived peptide variants of recombinant, synthetic or enzymatic origin.[Results] The recognition sequences of G12 and A1 antibodies were hexameric and heptameric epitopes, respectively. Although G12 affinity for the 33-mer was superior to A1, the sensitivity for gluten detection was higher for A1. This observation correlated to the higher number of A1 epitopes found in prolamins than G12 epitopes. Activation of T cell from gluten digested by glutenases decreased equivalently to the detection of intact peptides by A1 antibody. Peptide recognition of A1 included gliadin peptides involved in the both the adaptive and innate immunological response in celiac disease.[Conclusions] The sensitivity and epitope preferences of the A1 antibody resulted to be useful to detect gluten relevant peptides to infer the potential toxicity of food for celiac patients as well as to monitor peptide modifications by transglutaminase 2 or glutenases.This work was supported by the Asociación de Celiacos de Madrid (to Carolina Sousa), by the CTA (Corporación Tecnológica de Andalucía) and IDEA (Agencia de Innovación y Desarrollo de Andalucía) (to Angel Cebolla) and by grants BFU2007-64999 from Plan Nacional de Investigación científica, Desarrollo e Innovación tecnológica (Miniterio de Educación y Ciencia) and RICET-RD06/0021-0014, Spain (to Manuel C. López). Belén Morón was supported by a fellowship from Consejo Andaluz de Colegios Oficiales de Farmacéuticos.Peer reviewe

Análisis de los genes de nodulación implicados en la sulfatación de la molécula señal de Rhizobium tropici CIAT899

R.tropici CIAT899 es un simbionte de amplio rango de hospedador capaz de producir nódulos fijadores de nitrógeno en numerosas leguminosas, y que se caracteriza tanto por ser muy tolerante a altas temperaturas como por mostrar un amplio espectro de tolerancia a pH ácidos y básicos. La interacción entre los rizobios y las leguminosas depende, en gran parte, del intercambio de señales químicas entre ambos simbiontes. Los estudios de la molécula señal de esta cepa demostraron la presencia de 16 tipos de moléculas diferentes, que podrían ser las responsables del amplio rango de hospedador que dicha cepa presenta. El amplio rango de hospedador de dicha bacteria es debido a su gran espectro de factores Nod. Esta bacteria contiene los genes responsables de la sulfatación, modificación que controla la nodulación en Leucaena. En este trabajo se localizaron y se aislaron los genes nodHPQ. Posteriormente, se estudio su organización y su modo de regulación indicando que estos genes pertenecen el operón nodABCSUIJ, transcribiéndose a partir de la caja de nodulación localizada corriente arriba del gen nodA. La expresión de dichos genes se realiza de un forma dependiente del gen regulador nodD1. Por otro lado, el resto de las copias de este gen presentan un actividad débil o nula como es el caso de los genes nodD3 y nodD5, o podrían presentar otras actividades tales como la represión descrita en el caso del gen nodD2 de Rhizobium sp. NGR234. Los genes nodHPQ podrían también presentar una expresión constitutiva independiente de la presencia de la presencia de los flavonoides. La doble promoción de los genes nodHPQ podría estar relacionada con la función de dichos genes ya que cada uno de los dos promotores permitiría su expresión en las condiciones adecuadas. De este modo la caja de nodulación controla su expresión en las condiciones simbióticas y la segunda zona promotora controla la expresión en otras condiciones aú