Inactivation of the Sinorhizobium fredii HH103 rhcJ gene abolishes nodulation outer proteins (Nops) secretion and decreases the symbiotic capacity with soybean.

It has been postulated that nodulation outer proteins (Nops) avoid effective nodulation of Sinorhizobium fredii USDA257 to nodulate with American soybeans. S. fredii HH103 naturally nodulates with both Asiatic (non-commercial) and American (commercial) soybeans. Inactivation of the S. fredii HH103 gene rhcJ, which belongs to the tts (type III secretion) cluster, abolished Nop secretion and decreased its symbiotic capacity with the two varieties of soybeans. S. fredii strains HH103 and USDA257, that only nodulates with Asian soybeans, showed different SDS-PAGE Nop profiles, indicating that these strains secrete different sets of Nops. In coinoculation experiments, the presence of strain USDA257 provoked a clear reduction of the nodulation ability of strain HH103 with the American soybean cultivar Williams. These results suggest that S. fredii Nops can act as either detrimental or beneficial symbiotic factors in a strain-cultivar-dependent manner. Differences in the flavonoid-mediated expression of rhcJ with respect to nodA were also detected. In addition, one of the Nops secreted by strain HH103 was identified as NopA.     

Reversion of thermic-shock effect on ram spermatozoa by adsorption of seminal plasma proteins revealed by partition in aqueous two-phase systems

Centrifugal counter-current distribution (CCCD) in a dextran, Ficoll, poly(ethylene glycol) two-phase system was used to study the effect of seminal plasma proteins on the partition behaviour of ram spermatozoa exposed to thermal shock. Ram spermatozoa freed from seminal plasma by a ‘swim-up’ procedure were submitted to thermal shock and fractionated by CCCD. Cell viability decreased from 68% to 18% after the treatment, showing a slight displacement of the cells from the right (where a higher enrichment of live cells is found) to the centre of the profile. A change of the distribution profile was shown in the presence of either ram or bull seminal plasma. Bull seminal plasma was able to move the profile to the right, whereas ram seminal plasma increased the proportion of cells with enhanced affinity for the lower dextran-rich phase. Plasma proteins isolated from both seminal plasmas moved the profile to the right. In addition, cell viability rose to 48% after the CCCD run in the presence of ram plasma proteins. This restoring effect was lost when ram plasma proteins were thermally denatured. Bovine serum albumin was not only unable to move the profile to the right but even promoted displacement of the profile to the left. This negative effect was also observed when proteins from bull seminal plasma were in the presence of protein-free ram seminal plasma. However, proteins isolated from ram seminal plasma still restored the profile in the presence of bull seminal plasma freed from proteins. The results presented here strongly suggest that seminal plasma proteins are absorbed by a spermatozoal surface previously exposed to thermic shock. These proteins would exert a highly specific protective effect on ram spermatozoa. In addition, in the ram seminal plasma there must be some factor which avoids this adsorption.     

A pyrF auxotrophic mutant of Sinorhizobium fredii HH103 impaired in its symbiotic interactions with soybean and other legumes.

Transposon Tn5-Mob mutagenesis allowed the selection of a Sinorhizobium fredii HH103 mutant derivative (SVQ 292) that requires the presence of uracil to grow in minimal media. The mutated gene, pyrF, codes for an orotidine-5 - monophosphate decarboxylase (EC 4.1.1.23). Mutant SVQ 292 and its parental prototrophic mutant HH103 showed similar Nod-factor and lipopolysaccharide profiles. The symbiotic properties of mutant SVQ 292 were severely impaired with all legumes tested. Mutant SVQ 292 formed small ineffective nodules on Cajanus cajan and abnormal nodules (pseudonodules) unable to fix nitrogen on Glycine max (soybean), Macroptitlium atropurpureum, Indigofera tinctoria, and Desmodium canadense. It also did not induce any macroscopic response in Macrotyloma axillare roots. The symbiotic capacity of SVQ 292 with soybean was not enhanced by the addition of uracil to the plant nutritive solution.     

A Novel Lipidomic Strategy Reveals Plasma Phospholipid Signatures Associated with Respiratory Disease Severity in Cystic Fibrosis Patients

The aim of this study was to search for lipid signatures in blood plasma from cystic fibrosis (CF) patients using a novel MALDI-TOF-ClinProTools™ strategy, initially developed for protein analysis, and thin layer chromatography coupled to MALDI-TOF (TLC-MALDI). Samples from 33 CF patients and 18 healthy children were subjected to organic extraction and column chromatography separation of lipid classes. Extracts were analyzed by MALDI-TOF, ion signatures were compared by the ClinProTools™ software and by parallel statistical analyses. Relevant peaks were identified by LC-MSn. The ensemble of analyses provided 11 and 4 peaks differentially displayed in CF vs healthy and in mild vs severe patients respectively. Ten ions were significantly decreased in all patients, corresponding to 4 lysophosphatidylcholine (18∶0, 18∶2, 20∶3, and 20∶5) and 6 phosphatidylcholine (36∶5, O-38∶0, 38∶4, 38∶5, 38∶6, and P-40∶1) species. One sphingolipid, SM(d18∶0), was significantly increased in all patients. Four PC forms (36∶3, 36∶5, 38∶5, and 38∶6) were consistently downregulated in severe vs mild patients. These observations were confirmed by TLC-MALDI. These results suggest that plasma phospholipid signatures may be able to discriminate mild and severe forms of CF, and show for the first time MALDI-TOF-ClinProTools™ as a suitable methodology for the search of lipid markers in CF.     

NopM and NopD are rhizobial nodulation outer proteins: identification using LC-MALDI and LC-ESI with a monolithic capillary column

We have explored the potential of commercial polystyrene-divinylbenzene monolithic capillary nanoLC-MS/MS for identifying Sinorhizobium fredii HH103 nodulation outer proteins. Monolithic nanoLC with off-line MALDI-TOF/TOF and on-line ESI-q-oTOF is fast and robust, generating complementary data and offering high-confidence protein identifications from gel bands too weak for successful analysis using traditional approaches. This has allowed identification of two proteins not previously described as being type III-secreted in rhizobia, NopM and NopD.     

SAR study of 2,3,4,14b-tetrahydro-1H-dibenzo[b,f]pyrido[1,2-d][1,4]oxazepines as progesterone receptor agonists

We have developed a new class of progesterone receptor agonists having a tetracyclic dibenzo-oxazepine structure 1. In this paper, the synthesis and structure-activity relationships of this new class are described. This work led to the identification of potent progesterone agonists up to 1 nM activity. Substitution at positions 6, 7 and 1 has proven to be crucial for activity, indicating that probably these positions are involved in important interactions with the receptor.     

Effect of pH and soybean cultivars on the quantitative analyses of soybean rhizobia populations

Quantitative analyses of fast- and slow-growing soybean rhizobia populations in soils of four different provinces of China (Hubei, Shan Dong, Henan, and Xinjiang) have been carried out using the most probable number technique (MPN). All soils contained fast- (FSR) and slow-growing (SSR) soybean rhizobia. Asiatic and American soybean cultivars grown at acid, neutral and alkaline pH were used as trapping hosts for FSR and SSR strains. The estimated total indigenous soybean-rhizobia populations of the Xinjiang and Shan Dong soil samples greatly varied with the different soybean cultivars used. The soybean cultivar and the pH at which plants were grown also showed clear effects on the FSR/SSR rations isolated from nodules. Results of competition experiments between FSR and SSR strains supported the importance of the soybean cultivar and the pH on the outcome of competition for nodulation between FSR and SSR strains. In general, nodule occupancy by FSRs significantly increased at alkaline pH. Bacterial isolates from soybean cultivar Jing Dou 19 inoculated with Xinjiang soil nodulate cultivars Heinong 33 and Williams very poorly. Plasmid and lipopolysaccharide (LPS) profiles and PCR-RAPD analyses showed that cultivar Jing Dou 19 had trapped a diversity of FSR strains. Most of the isolates from soybean cultivar Heinong 33 inoculated with Xinjiang soil were able to nodulate Heinong 33 and Williams showed very similar, or identical, plasmid, LPS and PCR-RAPD profiles. All the strains isolated from Xinjiang province, regardless of the soybean cultivar used for trapping, showed similar nodulation factor (LCO) profiles as judged by thin layer chromatographic analyses. These results indicate that the existence of soybean rhizobia sub-populations showing marked cultivar specificity, can affect the estimation of total soybean rhizobia populations indigenous to the soil, and can also affect the diversity of soybean rhizobial strains isolated from soybean nodules.     

Sinorhizobium fredii HH103 does not strictly require KPS and/or EPS to nodulate Glycyrrhiza uralensis, an indeterminate nodule-forming legume

The Sinorhizobium fredii HH103 rkp-1 region, which is involved in capsular polysaccharide (KPS) biosynthesis, is constituted by the rkpU, rkpAGHIJ, and kpsF3 genes. Two mutants in this region affecting the rkpA (SVQ536) and rkpI (SVQ538) genes were constructed. Polyacrylamide gel electrophoresis and (1)H-NMR analyses did not detect KPS in these mutants. RT-PCR experiments indicated that, most probably, the rkpAGHI genes are cotranscribed. Glycine max cultivars (cvs.) Williams and Peking inoculated with mutants SVQ536 and SVQ538 showed reduced nodulation and symptoms of nitrogen starvation. Many pseudonodules were also formed on the American cv. Williams but not on the Asiatic cv. Peking, suggesting that in the determinate nodule-forming S. fredii-soybean symbiosis, bacterial KPS might be involved in determining cultivar-strain specificity. S. fredii HH103 mutants unable to produce KPS or exopolysaccharide (EPS) also showed reduced symbiotic capacity with Glycyrrhiza uralensis, an indeterminate nodule-forming legume. A HH103 exoA-rkpH double mutant unable to produce KPS and EPS was still able to form some nitrogen-fixing nodules on G. uralensis. Thus, here we describe for the first time a Sinorhizobium mutant strain, which produces neither KPS nor EPS is able to induce the formation of functional nodules in an indeterminate nodule-forming legume.     

Behaviour of a sym plasmid from Rhizobium 'hedysari' in different Rhizobium species

Abstract The symbiotic plasmid pRHc1J of Rhizobium 'hedysari' has been transferred to different Rhizobium species. It expression and incompatibility with the recipient resident plasmids as well as the effect of the host plants on the selection of Rhizobium symbiotic information has been studied. When the symbiotic plasmid pRHc1J was transferred to Nod⁺Fix⁺ Rhizobium species, it underwent specific deletions, either spontaneously or after the passage of transconjugants through plants, leading to the loss of some essential nod genes.