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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.

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.

Background [NiFe] hydrogenases are enzymes that catalyze the oxidation of hydrogen into protons and electrons, to use H 2 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 Δ 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 Strep Tag-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.

The establishment of a symbiotic interaction involves a signal exchange between the host legume (flavonoids) and the nitrogen-fixing rhizobia (nodulation factors (NFs)). Likewise, abiotic stress conditions, such as salinity and drought, strongly reduce the nodulation process, possibly affecting also the signal exchange. In this work we characterized the structure and biological activity of NFs produced by Bradyrhizobium japonicum USDA 138 under control, salt, and osmotic stress conditions. This strain is the most widely used in Argentine soybean culture; under control conditions, it produces a mixture of four types of NFs (V(C₁₆:₀,MeFuc), V(C₁₈:₁,MeFuc), IV(C₁₈:₁), and V(C₁₈:₁,Ac,MeFuc)). Interestingly, under stress conditions, this strain produces new types of NFs, one common for both stress conditions (V(C₁₆:₁,MeFuc)) and another one only present under salt stress (IV(C₁₈:₁,MeFuc)). All mixtures of NFs, extracted from control, salt, and osmotic stress conditions, showed biological activity in soybean plants, such as root hair deformation, and the radical application of purified NFs induced systemic differences in dry matter accumulation. The inoculation of soybean with genistein-induced bacteria cultured under both control and stress conditions had a positive effect on the number of nodules formed and in some cases on dry matter accumulation. These responses are not related to changes in chlorophyll fluorescence or greenness index.

[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.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.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 ΔhupF 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.RESULTSHupF 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 ΔhupF 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.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.CONCLUSIONSThe 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.

The root nodule bacterium Rhizobium tropici strain CIAT899 is highly stress resistant. It grows under acid conditions, in large amounts of salt, and at high osmotic pressure. An earlier study reported a substantial qualitative and quantitative effect of acid stress on the biosynthesis of Nod factors. The aim of the present work was to investigate the effect of high salt (NaCl) concentrations, another common stress factor, on Nod factor production. For this purpose, thin-layer chromatography, HPLC and MS analyses were carried out. The expression of nodulation genes was also studied using a nodPlacZ fusion. High concentrations of sodium enhanced nod gene expression and Nod factor biosynthesis. The effect is sodium specific because high potassium or chloride concentrations did not have this effect. Under salt stress conditions, 46 different Nod factors were identified in a CIAT899 culture, compared with 29 different Nod factors under control conditions. Only 15 Nod factor structures were common to both conditions. Under salt stress conditions, 14 different new Nod factor structures were identified that were not observed as being produced under neutral or acid conditions. The implications of our results are that stress has a great influence on Nod factor biosynthesis and that new, very interesting regulatory mechanisms, worth investigating, are involved in controlling Nod factor biosynthesis

In Morocco, storage reservoirs are particular systems of water supply in rural areas. These reservoirs are fed by rainwater and/or directly from the river through opened channels, and are used without any treatment as a drinking water by the surrounding population. This study was conducted to evaluate the bacterial contamination of drinking water reservoirs in this rural area, using a molecular approach studying the 16S-rDNA bacterial diversity in water, via Polymerase Chain Reaction and Denaturing Gradient Gel Electrophoresis (PCR-DGGE) technique. The application of PCR-DGGE techniques on stored water in the reservoirs showed a high bacterial diversity, including pathogens, namely Salmonella sp., E.coli, Sphingomonas spp. and Aeromonas sp., which indicated a high risk of infection for the user population. Comparative cluster analyses of the DNA based fingerprints revealed the six studied reservoirs according a gradient accumulation of bacterial contaminants from upstream to downstream. The molecular approach in this study gives a very helpful tool to confirm without any doubt the bacterial contamination of drinking water.

España, Ministerio de Ciencia y tecnología a BIO2010-15301

Rhizobium tropici CIAT899 is a tropical symbiont able to nodulate various legumes such as Leucaena, Phaseolus, and Macroptilium. Broad host range of this species is related to its Nod factors wide spectrum. R. tropici contains Nod factors sulphation nod genes, nodHPQ genes, which control nodulation efficiency in Leucaena. To study nodHPQ regulation, we carried out different interposon insertions in its upstream region. One of these generated interruptions, nodI mutant produced nonsulphated Nod factors suggesting a possible dependence of these genes on nodI upstream region. Moreover, analysis results of lacZ transcriptional fusions with these genes in symbiotic plasmid showed dependence of these genes on NodD protein. In order to determine nodHPQ organization, we studied the effect of interposon insertion upstream of each lacZ transcriptional fusion, and the data obtained was used to indicate that nodHPQ belong to the nodABCSUIJ operon. However, comparison between nodP::lacZ beta-galactosidase activity in the symbiotic plasmid and in the pHM500 plasmid (containing nodHPQ genes) suggested constitutive expression in free living, and flavonoid inducible expression in symbiotic conditions. Constitutive nodHPQ expression may play a role in bacterial house-keeping metabolism. On the other hand, the transference of R. tropici nodHPQ genes to other rhizobia that do not present sulphated substitutions demonstrated that NodH protein sulphotransference is specific to C6 at the reducing end.

Rhizobium tropici CIAT899 is a tropical symbiont able to nodulate various legumes such as Leucaena, Phaseolus, and Macroptilium. Broad host range of this species is related to its Nod factors wide spectrum. R. tropici contains Nod factors sulphation nod genes, nodHPQ genes, which control nodulation efficiency in Leucaena. To study nodHPQ regulation, we carried out different interposon insertions in its upstream region. One of these generated interruptions, nodI mutant produced nonsulphated Nod factors suggesting a possible dependence of these genes on nodI upstream region. Moreover, analysis results of lacZ transcriptional fusions with these genes in symbiotic plasmid showed dependence of these genes on NodD protein. In order to determine nodHPQ organization, we studied the effect of interposon insertion upstream of each lacZ transcriptional fusion, and the data obtained was used to indicate that nodHPQ belong to the nodABCSUIJ operon. However, comparison between nodP::lacZ β-galactosidase activity in the symbiotic plasmid and in the pHM500 plasmid (containing nodHPQ genes) suggested constitutive expression in free living, and flavonoid inducible expression in symbiotic conditions. Constitutive nodHPQ expression may play a role in bacterial house-keeping metabolism. On the other hand, the transference of R. tropici nodHPQ genes to other rhizobia that do not present sulphated substitutions demonstrated that NodH protein sulphotransference is specific to C6 at the reducing end.Key words: Nod factors, nodHPQ genes, Rhizobium tropici, nod-box.