PCR primers based on different portions of insertion elements can assist phylogeny studies, strain fingerprinting and species identification in rhizobia.

Using the sequence of an insertion element originally found in Rhizobium sullae, the nitrogen-fixing bacterial symbiont of the legume Hedysarum coronarium, we devised three primer pairs (inbound, outbound and internal primers) for the following applications: (a) tracing genetic relatedness within rhizobia using a method independent of ribosomal inheritance, based on the presence and conservation of IS elements; (b) achieve sensitive and reproducible bacterial fingerprinting; (c) enable a fast and unambiguous detection of rhizobia at the species level. In terms of taxonomy, while in line with part of the 16S rRNA gene- and glutamine synthetase I-based clustering, the tools appeared nonetheless more coherent with the actual geographical ranges of origin of rhizobial species, strengthening the European-Mediterranean connections and discerning them from the asian and american taxa. The fingerprinting performance of the outward-pointing primers, designed upon the inverted repeats, was shown to be at least as sensitive as BOX PCR, and to be functional on a universal basis with all 13 bacterial species tested. The primers designed on the internal part of the transposase gene instead proved highly species-specific for R. sullae, enabling selective distinction from its most related species, and testing positive on every R. sullae strain examined, fulfilling the need of PCR-mediated species identification. A general use of other IS elements for a combined approach to rhizobial taxonomy and ecology is proposed

Characterization of endophytic and symbiotic bacteria within plants of the endemic association Centaureetum horridae Mol.

We investigated the internal association of bacteria with Astragalus terraccianoi and Centaurea horrida, two endemic plants of the Mediterranean islands, forming the phytosociological association Centaureetum horridae, typical of windswept cliffs on the rocky shores of Asinara (Sardinia, Italy) and other limited locations. Sampling occurred in the protected natural park of the Asinara island. Roots and stems of the two plants and the root nodules of A. terraccianoi were surface sterilized in order to remove external and rhizospheric microbiota and to subsequently isolate the culturable bacterial communities. Plate counts revealed densities of endophytes between 3.7 7 102 and 2.8 7 104 colony forming units per gram of fresh weight. 16S rDNA sequencing revealed the occurrence of bacteria displaying high similarity with Actinobacterium sp., Paenibacillus sp., Rhizobium sp., Methylobacterium sp., Pedobacter panaciterrae, Aerococcus viridans, Stenotrophomonas rhizophila, Bacillus sporothermodurans, Bacillus pumilus, Bacillus simplex, Bacillus flexus, Streptomyces ciscaucasicus and Dyella sp. The putative nitrogen-fixing rhizobium symbiont of A. terraccianoi was identified for the first time. It turned out to belong to the slow-growing Bradyrhizobium genus and to share a 97% similarity with Bradyrhizobium canariense. It was found to be nonculturable and to coexist in nodules with a number of different endophytes

Wild legume root nodules as a potential reservoir for human pathogenic bacteria.

A previous finding by our group (Benhizia et al., 2004) shows that root nodules from wild legumes, besides their natural rhizobium symbionts, can host and multiply bacteria belonging to species pathogenic to humans. These include Enterobacter cloacae, Enterobacter kobei, Escherichia vulneris, Leclercia adecarboxylata, Pantoea agglomerans. As these taxa were repeatedly found in nodules from three plant species, differing by habitat ecophysiology, and harvested in independent natural sites which are spaced apart up to 150 Km from each other, we believe that the phenomenon can be a general feature and have potentially significant impacts for the epidemiology of bacteria of clinical interest. In the sole Italian territory nearly four hundred species of wild leguminous plants are known, whose microbiological interactions are largely unknown. These plants can nevertheless develop abundant root nodules, which are optimal sites for bacterial multiplication. Wild legume distribution can span over a series of habitats, ranging from urban-synanthropic, to agricultural, and to the majority of natural habitats. In light of the above findings, yielding five Enterobacterial taxa of potential danger to humans from the analysis of only three species of wild plants, one could envisage the biomass of wild legumes as possible strategic niche for the survival and active multiplication of clinical pathogens in hosts alternative to mammals

Reinterpreting quorum sensing as positional sensing in bacterial communication

Background and Aims The phenomenon of bacterial cell-to-cell communication via n-acyl homoserine lactone molecules was examined in a simple series of direct microscopy tests aimed at verifying the postulates of the current quorum sensing working model. Methods Using a wild type strain of Rhizobium leguminosarum, we first devised a signal production system in which cell number, cell density and cell distance could be varied at leisure while maintaining the physiological status and preventing further cell divisions. The model was subsequently verified by epifluorescent microscopy in a tri-dimensional set up in LB agar, using an rfp-tagged AHL producing strain of Pseudomonas aeruginosa and a sensor Escherichia coli bearing a gfp-fused AHL reporter gene. Results Data obtained by measuring 697 individual cell-to-cell communication distances using the CMEIAS image analysis software showed that indeed quorum sensing can be reduced to an interaction involving as few as two individual cells, with a mean calling distance of 37.4 um and a maximum calling distance extended as far as 123.6 um. Direct microscopy showed that a single bacterium can produce sufficient signal in situ to activate a population of 180 bacterial cells. Conclusions The results indicate that quorum sensing does not comply with the paradigm of a mechanism simply depending on cell density. A novel concept of positionally-releated sensing, via mixing concentration gradients, is presented

Molecular signals in Pseudomonas tolaasii/Pleurotus eryngii host/pathogen interactions;

P. tolaasii strain P12 has been found in association with brown blotch disease on Pleurotus eryngii, one of the more extensively cultivated mushrooms in the Mediterranean area. Bacterial population density represents a key factor as the infected basidiocarps develop disease symptoms when the pathogen concentration is higher than 104 cfu/g d.w. Virulence of P. tolaasii, mainly due to tolaasin production, may be regulated by quorum sensing (QS) cell-density-dependent regulatory mechanism related to N-acyl homoserine lactones (AHLs). The capacity of P12 to produce AHLs has been investigated. Very low signals related to the production of AHLs were obtained in P12 and other five P. tolaasii strains. Moreover, the capacity to enter the viable but not culturable (VBNC) state was observed under low nutrient anaerobic conditions. As in others bacteria the ability to enter the VBNC state has been associated to the production of QS signals, the above conditions were adopted for QS signal production experiments. Data obtained suggest that specific conditions must be needed to activate QS genes in P12. The involvement of these specific factors in the occurrence of the disease has to be elucidated

Shovel roots: a unique stress-avoiding developmental strategy of the legume plant Hedysarum coronarium L.

Hedysarum coronarium (sulla) is a legume native to the Mediterranean basin, known for its broad tolerance to various environmental stresses, and its ability to thrive without signs of chlorosis when growing in arid and alkaline soils up to pH 9.6. A unique but poorly known morphological feature of its root system is the production of "shovels", modified lateral roots that acquire a curved and flattened shape. A combined structural and functional analysis was undertaken to define the nature and role of the shovel roots using various microscopy techniques, histochemical stains, STEM - energy dispersive X-ray microanalysis, infrared spectroscopy, and plant cultivation in different conditions. We found that sulla displays remarkable unique rhizosphere-buffering properties at both ends of the pH scale, and that shovels act as efficient calcium-absorbing organs that accumulate this cation intracellularly as insoluble crystalline salts. Such bioaccumulation results in a localized depletion of CaCO(3) from the soil. As a consequence of this removal of the pivotal carbonate buffering system, the iron-solubilizing acidification activities of the roots can become effective. Further tests revealed that the factor triggering shovel development is exposure of roots to iron oxide. This signal, reporting at once both iron presence and alkalinity, assures the availability of iron nutrient reserves upon acidification of the local microenvironment surrounding the roots. These findings, besides casting light on a novel and unique botanical phenomenon, offer the potential to exploit sulla's model and genes for the improvement of other crops to sustain productivity in a scenario of climate warming and increasing desertification

Long term evaluation of field-released genetically modified rhizobia

This is the report of the first open field release of genetically modified microorganisms (GMMs) in Italy. It covers ten years of monitoring, and follows in-field GMM dynamics from strain release to disappearance below detection limits, as well as assessment of impact on resident microorganisms. The bacteria released belong to the nitrogen fixing legume endosymbiont Rhizobium leguminosarum bv. viciae, and were engineered with non-agronomically-proficient traits, in order to assess their behavior and fate without GMM-specific positive feedback from the plant. A DNA cassette containing mercury resistance and ss-galactosidase genes was introduced in either plasmid-borne or chromosomally integrated versions, in order to test the resulting strain stability. A synthetic promoter was used to drive the lacZ gene, conferring high catabolic activity to the GMM. Two different wild-type Rhizobium backgrounds were tested, comparing a non-indigenous vs. an indigenous, highly competitive strain. The latter had much greater persistence, since it was able to survive and establish at technically detectable levels for over four years after release. Selection factors, such as reiterated presence of the plant host, or lactose substrate supply, enhanced long-term survival to different extents. The lactose treatment showed that even a single trophic supplementation can surpass the benefits of symbiotic interaction for a period of several years. Concerning impact, the GMMs did not alter substantially the other soil community general microbiota. However, there were some significant differences in microbiota as a consequence of the Rhizobium inoculation. This effect was observed with either the WT or GMM, and was more evident in the release of the indigenous Rhizobium. Moreover, as the indigenous GMM had its parental, dominant wild-type in the same soil, it was possible to evaluate to what extent the GMM version could result in parent displacement ("self-impact"), and how much the two rhizobia would additively contribute to nodulation