Draft genome sequence of the nitrogen-fixing Rhizobium sullae type strain IS123^T focusing on the key genes for symbiosis with its host Hedysarum coronarium L.

© 2017 Sablok, Rosselli, Seeman, van Velzen, Polone, Giacomini, La Porta, Geurts, Muresu and Squartini. The prominent feature of rhizobia is their molecular dialogue with plant hosts. Such interaction is enabled by the presence of a series of symbiotic genes encoding for the synthesis and export of signals triggering organogenetic and physiological responses in the plant. The genome of the Rhizobium sullae type strain IS123T nodulating the legume Hedysarum coronarium, was sequenced and resulted in 317 scaffolds for a total assembled size of 7,889,576 bp. Its features were compared with those of genomes from rhizobia representing an increasing gradient of taxonomical distance, from a conspecific isolate (Rhizobium sullae WSM1592), to two congeneric cases (Rhizobium leguminosarum bv. viciae and Rhizobium etli) and up to different genera within the legume-nodulating taxa. The host plant is of agricultural importance, but, unlike the majority of other domesticated plant species, it is able to survive quite well in the wild. Data showed that that the type strain of R. sullae, isolated from a wild host specimen, is endowed with a richer array of symbiotic genes in comparison to other strains, species or genera of rhizobia that were rescued from domesticated plant ecotypes. The analysis revealed that the bacterium by itself is incapable of surviving in the extreme conditions that its host plant can tolerate. When exposed to drought or alkaline condition, the bacterium depends on its host to survive. Data are consistent with the view of the plant phenotype as the primary factor enabling symbiotic nitrogen fixing bacteria to survive in otherwise limiting environments

Chemical Elemental Distribution and Soil DNA Fingerprints Provide the Critical Evidence in Murder Case Investigation

Background: The scientific contribution to the solution of crime cases, or throughout the consequent forensic trials, is a crucial aspect of the justice system. The possibility to extract meaningful information from trace amounts of samples, and to match and validate evidences with robust and unambiguous statistical tests, are the key points of such process. The present report is the authorized disclosure of an investigation, carried out by Attorney General appointment, on a murder case in northern Italy, which yielded the critical supporting evidence for the judicial trial. Methodology/Principal Findings: The proportional distribution of 54 chemical elements and the bacterial community DNA fingerprints were used as signature markers to prove the similarity of two soil samples. The first soil was collected on the crime scene, along a corn field, while the second was found in trace amounts on the carpet of a car impounded from the main suspect in a distant location. The matching similarity of the two soils was proven by crossing the results of two independent techniques: a) elemental analysis via inductively coupled plasma mass spectrometry (ICP-MS) and optical emission spectrometry (ICP-OES) approaches, and b) amplified ribosomal DNA restriction analysis by gel electrophoresis (ARDRA). Conclusions: Besides introducing the novel application of these methods to forensic disciplines, the highly accurate level of resolution observed, opens new possibilities also in the fields of soil typing and tracking, historical analyses, geochemical surveys and global land mapping

Intercellular communication in bacteria nodulating plants of the family Leguminosae

In order to achieve a better understanding of the nodulation patterns of rhizobia in their symbiosis with legume hosts, and with the aim to examine their signalling behaviour in cell-to-cell communication, a series of experimental projects were carried out. In first instance the microbial inhabitants of 831 pea root nodules formed on nine plants, sown in different field soil parcels, were isolated and characterized by PCR-based electrophoretic fingerprinting using the BOXA1R primer. Band profiles were analyzed by GelComparII image analysis software converting differences into a numerical matrix yielding their similarity dendrogram in terms of genetic fingerprint distances. The level of strain-specific association with individual plant or soil plots has been assessed. As 85% of the profiles result singletons, having been found in only one nodule, the overall diversity of the site appears particularly high. Estimates of the total diversity at biovar level were obtained by nonparametric estimators pointing to a value over 1300 types. Such richness was compared with the much lower one recorded eight years earlier on the same plots and was put in relation with the repeated host cropping occurred in between. Moreover, the position of each nodule within the root apparatus, in terms of root rank order and distance from the crown, had been recorded in digitized images and the existence of topological and temporal patterns in each strain's nodulation process has been inspected. The fingerprinting quality of BOX-PCR in terms of reproducibility and sensitivity, was compared to that obtainable by other primers as ISRh1 outbound primers. The same fingerprint-characterized strains were screened for the production of Quorum Sensing signals consisting in short-medium- (C4-C8) and long- (C14) chained N-acyl homoserine lactones (AHLs) using, respectively, the two reporter systems: Chromobacterium violaceum CV026 (violacein pigment induction) and Rhizobium leguminosarum A34 (colony growth inhibition). The majority of the natural Rhizobium leguminosarum strains were found to be quorum-signalling positive. The occurrence of isolates negative to one or both phenotypes however shows that those traits are not absolute requirements for host nodulation. In a different study we examined the root nodule symbionts of eight species of wild legumes collected in Sardinia. Interestingly, unlike the case of cultivated legumes, the recovery on plates of the rhizobial occupant could not be obtained under any of the conditions used, while at the same time a number of different endophytic taxa were rescued and their taxonomic identity was determined by 16S nucleotide sequencing. By direct PCR analysis from the nodule tissue, we were also able to show the presence of the nonculturable rhizobia inside the same nodules. In parallel, other two studies were conducted. AHL-mediated quorum sensing communication was quantified at single cell resolution trough a red-fluorescing AHLproducing and a green-fluorescing AHL-sensor strain in a 3-dimensional system by using computer-assisted microscopy (CMEIAS). The average effective "calling distance" from the single cell producer capable of inducing the gfp-tagged reporter cells, resulted 46.8 ?m. Moreover, in relation to the possible involvement of AHL signals in different phenotypes, a series of plant-interacting strains, among which Rhizobium leguminosarum, were tested for their ability to maintain viability in stressful situations (nutrient and oxygen limitations). Some of the tested strains lost culturability in different of the imposed conditions. However viable cells could be detected by staining microscope-based techniques (BacLight®, acridine orange and CTC), demonstrating that the treated bacteria changed into a viable but nonculturable (VBNC) form; none of the AHL Quorum Sensing signals tested was effective in promoting the transition to the VBNC state nor in recovering cells to culturability, suggesting that the two phenotypical frameworks of QS and VBNC do not share signalling paths

PCR primers based on different portions of insertion elements can assist genetic relatedness 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

Use of antioxidant treatments to enhance culturability of rhizobial and non-rhizobial endophytes from nodules and other plant tissues

When rescuing either symbionts or other endophytic bacteria from internal plant tissues, including root nodules, and attempting their isolation and culturing on laboratory media, failure to obtain colonies is often experienced. This phenomenon applies particularly to the rhizobia from wild legume species which frequently result in non-culturable status (1), possibly as a consequence of a biochemical stress inherent to the isolation procedure. Other taxa can co-occupy nodules along with rhizobia, mostly belonging to the Bacillaceae, Pseudomonadaceae and Enterobacteriaceae families, which appear much less sensitive to the putative stress as their isolation from nodules, is apparently not impaired. We hypothesized that the limited culturability of rhizobia, compared to that of other types of nodule invaders, could be related to an oxidative stress caused by the histological disruption of plant tissues when surface-sterilized nodules are squashed for bacterial isolation. In order to test this hypothesis we isolated bacteria from nodules of two mediterranean wild legumes (Hedysarum spinosissimum and Tetragonolobus purpureus) by squashing nodules either in control phosphate buffer saline (PBS) or in PBS supplemented with scavenging systems apt to prevent damage from reactive oxygen species. The treatments included: an antioxidant cocktail (Glutathione, Sodium Ascorbate and EDTA) or an enzyme cocktail (Catalase, Peroxidase, Superoxide Dismutase). The mixtures were tested either as modified squashing buffers or as additions to the plates on which bacteria were spread after squashing in control PBS. The latter option allowed to distinguish whether the oxidative stress were due to immediate damage from plant-derived reactions or as a consequence of the transfer in carbon-rich agar media from the plate culturing stage. Some of the combinations yielded orders of magnitude-different increases of culturability in terms of colony counts and also allowed the cultivation of otherwise undetectable taxa. The coexistence of up to three different rhizobiaceae within the same nodules was observed, two of which of supposedly non-symbiotic nature (unable to re-induce nodule formation in axenic nodulation tests). Isolates were subsequently tested for tolerance to H2O2 in liquid cultures, which showed differences in comparison with the levels displayed by non-alphaproteobacterial endophytes. Ecological and physiological implications are discussed

Antioxidant treatments counteract the non-culturability of bacterial endophytes isolated from legume nodules

In many wild legumes, attempts to cultivate nodule bacteria fail. We hypothesized that the limited culturability could be related to injury from oxidative stress caused by disruption of plant tissues during isolation. To test that, we isolated bacteria from nodules of Hedysarum spinosissimum and Tetragonolobus purpureus using buffers supplemented with scavenging systems to prevent damage from reactive oxygen species (ROS). Treatments included the following: antioxidants (glutathione, ascorbate, EDTA) or enzymes (catalase, peroxidase, superoxide dismutase), tested either as modified squashing buffers or added in plates. Some combinations yielded dramatic increases of culturability. Different endophytes were found, including additional Rhizobiaceae that were not the primary symbiont and were unable to nodulate. Their H2O2 tolerance in broth culture showed differences consistent with the unequal culturability observed. In wild legumes species, ROS generation during extraction appears to be a major factor limiting microbiota isolation, and protocols presented here significantly improve the recovery of culturable bacterial endophytes from plants

Consequences of relative cellular positioning on quorum sensing and bacterial cell-to-cell communication

Cell-to-cell bacterial communication via diffusible signals is addressed and the conceptual framework in which quorum sensing is usually described is evaluated. By applying equations ruling the physical diffusion of the autoinducer molecules, one can calculate the gradient profiles that would occur either around a single cell or at the center of volumes of increasing size and increasing cell densities. Water-based matrices at 25 degrees C and viscous biofilms at colder temperatures are compared. Some basic consequences relevant for the field of microbial signalling arise. As regards induction, gradient-mixing dynamics between as little as two cells lying at a short distance appears to be sufficient for the buildup of a concentration reaching the known thresholds for quorum sensing. A straight line in which the highest concentrations occur is also created as a consequence of the gradient overlap geometry, providing an additional signal information potentially useful for chemotactic responses. In terms of whole population signalling, it is shown how the concentration perceived by a cell in the center is critically dependent not only on the cell density but also on the size of the biofilm itself. Tables and formulas for the practical prediction of N-acyl homoserine lactones concentrations at desired distances in different cell density biofilms are provided

Results of the analyses on the soil samples compared with two specimens of soil (CarR, CarL), found respectively on the right and left carpets of the suspect's car floor.

<p>a, b) ICP analysis of the content of 54 mineral elements. a): Box and whiskers synthetic representation of the variability of the 12 zones; b) Cluster analysis (single linkage, Euclidean distance) of the data; c) Amplified Ribosomal DNA Restriction Analysis (ARDRA) of the soil bacterial communities. The Neighbour Joining dendrogram resulting from Pearson correlation analysis of the combined three enzymes electrophoretic profiles is shown. The horizontal scale indicates the percent distance. In b) and c) sample replicates are included to show the degree of inter-replicate variability.</p