Non-culturable Rhizobia and diverse endophytic bacteria co-inhabiting wild legume nodules.

Analyses of the bacterial content of legume nodules does generally yield cultures of the symbiotic partner responsible of nodule induction, invasion and nitrogen fixation. Species involved in such interaction include examples among the alpha and beta-rhizobia. In the present survey, exploring the nodular content of several species of wild legumes in the Mediterranean basin, we witnessed a consistent occurrence of the following situation: the bacteria which could be isolated from surface- sterilized nodules do not represent the actual nodule-inducing microorganism but rather a variety of endophytic invaders. Their presence in the nodules appeared related to their more general and systemic distribution throughout the whole plant, in close analogy with the situation observed in many other herbaceous and woody species which host an array of on-pathogenic endophytic taxa. At the same time the presence and quantitative abundance of the real nodule symbiont in the wild legumes covered by our study could be demonstrated by 16S rRNA gene PCR and nucleotide sequencing, which revealed that, despite their failure to grow on plates, alpha-proteobacterial rhizobia do represent the bona-fide symbiont responsible of nodule organogenesis

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

Metabolic responses to long-term pharmacological inhibition of CB1-receptor activity in mice in relation to dietary fat composition

Background and objectives: The antiobesity effects of suppressed endocannabinoid signaling may rely, at least in part, on changes in lipid fluxes. As fatty acids exert specific effects depending on their level of saturation, we hypothesized that the dietary fatty acid composition would influence the outcome of treatment with a CB1-receptor antagonist (rimonabant). Methods: Mice were treated with rimonabant (10 mg kg-1 body weight per day) or vehicle while equicalorically fed either a low-fat diet (LF), a high-fat (HF) diet or an HF diet in which 10% of the saturated fatty acids (SFAs) were replaced by poly-unsaturated fatty acids (PUFA) from fish oil (FO). Food intake and body weight were registered daily. Indirect calorimetry was performed and feces were collected. After 3 weeks, mice were killed for blood and tissue collection. Results: Relative to the LF diet, the HF diet caused anticipated metabolic derangements, which were partly reversed by the HF/FO diet. The HF/FO diet, however, was most obesity-promoting despite inhibiting lipogenesis as indicated by low gene expression levels of lipogenic enzymes. On all three diets, rimonabant treatment improved metabolic derangements and led to significantly lower body weight gain than their respective controls. This latter effect appeared largest in the HF/FO group, but occurred without major changes in nutrient absorption and energy expenditure. Conclusion: The effects of chronic rimonabant treatment on body weight gain occurred irrespective of diet-induced changes in lipogenic activity, food intake and daily energy expenditure, and were, in fact, most pronounced in HF/FO mice. The effects of dietary PUFA replacement in an HF diet on expansion of adipose tissue might allow the favorable effects of dietary PUFA on dyslipidemia and hepatic steatosis. In light of other disadvantageous effects of weight gain, this might be a risky trade-off.