Coexistence of predominantly nonculturable rhizobia with diverse, endophytic bacterial taxa within nodules of wild legumes

A previous analysis showed that Gammaproteobacteria could be the sole recoverable bacteria from surface-sterilized nodules of three wild species of Hedysarum. In this study we extended the analysis to eight Mediterranean native, uninoculated legumes never previously investigated regarding their root-nodule microsymbionts. The structural organization of the nodules was studied by light and electron microscopy, and their bacterial occupants were assessed by combined cultural and molecular approaches. On examination of 100 field-collected nodules, culturable isolates of rhizobia were hardly ever found, whereas over 24 other bacterial taxa were isolated from nodules. None of these nonrhizobial isolates could nodulate the original host when reinoculated in gnotobiotic culture. Despite the inability to culture rhizobial endosymbionts from within the nodules using standard culture media, a direct 16S rRNA gene PCR analysis revealed that most of these nodules contained rhizobia as the predominant population. The presence of nodular endophytes colocalized with rhizobia was verified by immunofluorescence microscopy of nodule sections using an Enterobacter-specific antibody. Hypotheses to explain the nonculturability of rhizobia are presented, and pertinent literature on legume endophytes is discussed.     

TNF cytokine family: More BAFF-ling complexities

Recent studies on BAFF, a member of the tumor necrosis factor family, and the discovery of a new BAFF receptor have revealed that this ligand-receptor pair is essential for B-cell survival and differentiation, holding promise for a better understanding and treatment of some autoimmune diseases and lymphomas.     

Gamma proteobacteria can nodulate legumes of the genus Hedysarum

The bacteria hosted in the root nodules of the three Mediterranean wild legume species Hedysarum carnosum, Hedysarum spinosissimum subsp. capitatum, and Hedysarum pallidum, growing in native stands in different habitats in Algeria were isolated. Bacteria were recovered on yeast-mannitol-agar or on minimal media from a total of 52 nodules. Isolates were analyzed by Amplified Ribosomal DNA Restriction Analysis (ARDRA) using the enzyme CfoI, and further sorted by RAPD fingerprinting. A total of ten different types were found and their amplified 16S rDNA was sequenced and compared to databases. The BLAST alignment indicates that all the species whose sequences share 98 to 100% identity to the bacteria found in these nodules belong to the class Gammaproteobacteria and include Pantoea agglomerans, Enterobacter kobei, Enterobacter cloacae, Leclercia adecarboxylata, Escherichia vulneris, and Pseudomonas sp. No evidence of any rhizobial-like sequence was found even upon amplifying from the bulk of microbial cells obtained from the squashed nodules, suggesting that the exclusive occupants of the nodules formed by the three plants tested are members of the orders Enterobacteriales or Pseudomonadales. This is the first report of Gammaproteobacteria associated with legume nodules. Despite the presence of the related crop plant Hedysarum coronarium, specifically nodulated by Rhizobium sullae, these three Hedysarum species demonstrate to have undergone a separate path in terms of endophytic interactions with bacteria. An hypothesis to account for differences between the symbiotic relationships engaged by man-managed legumes, and those found in plants whose ecology is independent from human action, is discussed.     

Characterization of bacteria associated with nodules of two endemic legumes of Algeria,Hedysarum naudinianum and H. perrauderianum

The root nodules of two wild legume species endemic to Algeria, Hedysarum naudinianum and He. perrauderianum, were investigated with regard to their anatomy and histology, and the identity of the associated bacteria. Both plants were found to form root nodules with regular features and well infected by rod-shaped bacteria. The culturable fraction of bacteria that could be obtained from surface-sterilized nodules included a prevailing presence of Enterobacteriaceae having 100 % 16S rDNA sequence identity with both Enterobacter cloacae and E. ludwigii. In H. perrauderianum, this taxon was the sole cultured isolate, while from H. naudinianum we also found Bacillus, Lactobacillus, Staphylococcus, Rothia, and isolates that were 100 % identical to Corynebacterium pseudodiphthericum, which is known to be an agent of respiratory and cardiac infections in humans. Whereas no culturable rhizobia and alike could be obtained on plates, PCR-based culture-independent approaches revealed in both plants the presence of a Mesorhizobium sp., which in H. perrauderianum was identical to isolates nodulating other legumes from Africa, European Mediterranean countries, and Asia, while in H. naudinianum it bore a single nucleotide polymorphism which is so far unique for any observed mesorhizobia. Data from the microsymbionts appear to suggest interesting clues to interpret the evolutionary ecology of their host plants.     

Rheological and light scattering properties of flaxseed polysaccharide aqueous solutions

Polysaccharides isolated from flaxseed meals using ethanol consisted of a soluble ( approximately 7.5% w/w) and an insoluble fraction (2% w/w). The soluble fraction was dialyzed in various salt concentrations and characterized using viscometry and light scattering techniques. Observations using a size-exclusion column coupled to a multiangle laser light scattering (SEC-MALLS) revealed three molecular weight fractions consisting of a small amount ( approximately 17%) of large molecular weight species (1.0 x 10(6)) and a large amount ( approximately 69%) of small molecular weight species (3.1 x 10(5) Da). Dynamic light scattering measurements indicated the presence of very small molecules (hydrodynamic radius approximately 10 nm) and a very large molecular species (hydrodynamic radius in excess of 100 nm); the latter were probably aggregates. The intrinsic viscosity, [eta], of the polysaccharide in Milli-Q water was 1030 +/- 20 mL/g. The viscosity was due largely to the large molecular weight species since viscosity is influenced by the hydrodynamic volume of molecules in solution. The Smidsrod parameter B obtained was approximately 0.018, indicating that the molecules adopted a semi-flexible conformation. This was also indicated by the slope ( approximately 0.56) from the plot of root-mean-square (RMS) radius versus molar mass (M(w)).     

TNF deficiency fails to protect BAFF transgenic mice against autoimmunity and reveals a predisposition to B cell lymphoma

TNF is well characterized as a mediator of inflammatory responses. TNF also facilitates organization of secondary lymphoid organs, particularly B cell follicles and germinal centers, a hallmark of T-dependent Ab responses. TNF also mediates defense against tumors. We examined the role of TNF in the development of inflammatory autoimmune disorders resembling systemic lupus erythematosus and Sj?gren's syndrome induced by excess B cell-activating factor belonging to the TNF family (BAFF), by generating BAFF-transgenic (Tg) mice lacking TNF. TNF(-/-) BAFF-Tg mice resembled TNF(-/-) mice, in that they lacked B cell follicles, follicular dendritic cells, and germinal centers, and have impaired responses to T-dependent Ags. Nevertheless, TNF(-/-) BAFF-Tg mice developed autoimmune disorders similar to that of BAFF-Tg mice. Disease in TNF(-/-) BAFF-Tg mice correlates with the expansion of transitional type 2 and marginal zone B cell populations and enhanced T-independent immune responses. TNF deficiency in BAFF-Tg mice also led to a surprisingly high incidence of B cell lymphomas (>35%), which most likely resulted from the combined effects of BAFF promotion of neoplastic B cell survival, coupled with lack of protective antitumor defense by TNF. Thus, TNF appears to be dispensable for BAFF-mediated autoimmune disorders and may, in fact, counter any proneoplastic effects of high levels of BAFF in diseases such as Sj?gren's syndrome, systemic lupus erythematosus, and rheumatoid arthritis.     

Viscometric and static light scattering studies on an exopolysaccharide produced by Lactobacillus delbrueckii subspecies bulgaricus NCFB 2483

The rheological properties and molecular parameters of a purified exopolysaccharide (EPS) produced by a ropy strain of Lactobacillus delbrueckii subsp. bulgaricus NCFB 2483 were investigated. Using capillary viscometry, an intrinsic viscosity of 2,013 mL/g was obtained. The flow curves were fitted by both the Carreau and the Cross equations for shear-thinning fluids, with the Carreau equation giving a better fit. The Cross equation fitted fairly well the plot of reduced viscosity as a function of reduced shear rate with an exponent value (1 - n) of approximately 0.76, typical of random coil polymers. Furthermore, the concentration dependence of the viscosity plot showed a gradient of approximately 1.1 in the dilute regime and 3.3 in the semidilute regime. Molecular parameters were obtained using a multiangle laser light scattering technique. The 2483 EPS molecules had a weight-average molar mass of approximately 2 x 10(6) Da and a z-average root mean square radius (RMS) of approximately 151 nm. From the light scattering data, the bacterial EPS was also found to have a low polydispersity index (approximately 1.15) and adopt a random coil conformation.