Feminising Wolbachia disrupt Armadillidium vulgare insulin-like signalling pathway.

The endosymbiont Wolbachia feminises male isopods by making them refractory to the insulin-like masculinising hormone, which shunts the autocrine development of the androgenic glands. It was, therefore, proposed that Wolbachia silences the IR receptors, either by preventing their expression or by inactivating them. We describe here the two IR paralogs of Armadillidium vulgare. They displayed a conventional structure and belonged to a family widespread among isopods. Av-IR1 displayed an ubiquist expression, whereas the expression of Av-IR2 was restricted to the gonads. Both were constitutively expressed in males and females and throughout development. However, upon silencing, altered gland physiology and gene expression therein suggested antagonistic roles for Av-IR1 (androinhibiting) and Av-IR2 (androstimulating). They may function in tandem with regulating neurohormones, as a conditional platform that conveys insulin signalling. Wolbachia infection did not alter their expression patterns: leaving the IRs unscathed, the bacteria would suppress the secretion of the neurohormones, thus inducing body-wide IR deactivation and feminisation. Adult males injected with Wolbachia acquired an intersexed physiology. Their phenotypes and gene expressions mirrored the silencing of Av-IR1 only, suggesting that imperfect feminisation stems from a flawed invasion of the androstimulating centre, whereas in fully feminised males invasion would be complete in early juveniles. TAKE AWAY: Two antagonistic Insulin Receptors were characterised in Armadillidium vulgare. The IRs were involved in androstimulating and androinhibiting functions. Wolbachia-induced feminisation did not prevent the expression of the IRs. Imperfectly feminised intersexes phenocopied the silencing of Av-IR1 only. Wolbachia would deactivate the IRs by suppressing neurosecretory co-factors.

Lignocellulose degradation in isopods: new insights into the adaptation to terrestrial life.

BACKGROUND: Isopods constitute a particular group of crustaceans that has successfully colonized all environments including marine, freshwater and terrestrial habitats. Their ability to use various food sources, especially plant biomass, might be one of the reasons of their successful spread. All isopods, which feed on plants and their by-products, must be capable of lignocellulose degradation. This complex composite is the main component of plants and is therefore an important nutrient source for many living organisms. Its degradation requires a large repertoire of highly specialized Carbohydrate-Active enZymes (called CAZymes) which are produced by the organism itself and in some cases, by its associated microbiota. The acquisition of highly diversified CAZymes could have helped isopods to adapt to their diet and to their environment, especially during land colonization. RESULTS: To test this hypothesis, isopod host CAZomes (i.e. the entire CAZyme repertoire) were characterized in marine, freshwater and terrestrial species through a transcriptomic approach. Many CAZymes were identified in 64 isopod transcriptomes, comprising 27 de novo datasets. Our results show that marine, freshwater and terrestrial isopods exhibit different CAZomes, illustrating different strategies for lignocellulose degradation. The analysis of variations of the size of CAZy families shows these are expanded in terrestrial isopods while they are contracted in aquatic isopods; this pattern is probably resulting from the evolution of the host CAZomes during the terrestrial adaptation of isopods. We show that CAZyme gene duplications and horizontal transfers can be involved in adaptive divergence between isopod CAZomes. CONCLUSIONS: Our characterization of the CAZomes in 64 isopods species provides new insights into the evolutionary processes that enabled isopods to conquer various environments, especially terrestrial ones.

IGFBP-rP1, a strongly conserved member of the androgenic hormone signalling pathway in Isopoda.

The first protein which has been described to interact with the malacostracan Androgenic Gland Hormone (AGH) is a binding protein called IGFBP-rP1. It has been identified and studied in several species of decapods, in which its interaction with the masculinizing hormone and its expression patterns have been established in several ways. However, this protein remains uncharacterised to date in the other malacostracan orders, like Amphipoda and Isopoda, although they were historically the first ones in which the androgenic gland and the corresponding hormone were respectively described. In this article, we identified the IGFBP-rP1 of isopods and established its implication in the pathway of the AGH with a silencing approach in the model species Armadillidium vulgare. We also showed that this gene is expressed in all the tissues of males and females, with a similar pattern in animals infected with Wolbachia, a feminizing endosymbiont of several isopod species. The expression pattern did not differ during the development of uninfected and infected animals either. We finally studied the evolution of the IGFBP-rP1 in 68 isopod species, looking for conserved motifs and evidence of natural selection. Altogether, our results showed that this gene is constitutively expressed and strongly conserved in isopods, in which it likely constitutes a key element of the insulin/IGF signalling pathway. However, we also illustrated that IGFBP-rP1 is not sufficient on its own to explain the different developmental paths taken by the males and the females or feminized genetic males.

Divergent evolution and clade-specific duplications of the Insulin-like Receptor in malacostracan crustaceans.

The Insulin-like Receptors (IRs) are an important protein family, represented by three members in vertebrates, two of which are well-known for their implication in metabolism (Insulin Receptor) and growth (IGF Receptor). In contrast, little is known about these receptors in invertebrates, in which a single gene generally exists except for a part of insects and other occasional species-specific duplications. In this study, we used publicly available sequences as well as de novo assembled transcriptomes to investigate the IR evolution in malacostracan crustaceans, animals in which the Insulin/IGF pathway is known to be implicated in sexual development through the androgenic gland hormone. We described the evolutionary divergence of malacostracan IRs compared to all the other metazoan sequences, including other pancrustaceans. We also demonstrated two well conserved duplications of IRs: one specific to the whole malacostracan class, another one specific to the decapod order. The potential implications for malacostracan biology are discussed.

Characterization of biofilm formation in natural water subjected to low-frequency electromagnetic fields.

Electromagnetic field (EMF) treatment has proven to be effective against mineral scaling in water systems. Therefore, it should be assessed for the treatment of other deposits such as biofilms. In this study, a commercial device producing low-frequency EMF (1-10 kHz) was applied to a reactor fed with natural water for 45 days. The treatment promoted the concentration of microorganisms in suspension and limited the amount of sessile microorganisms in the biofilm, as determined by the measurement of total DNA, qPCR and microscopy. The structure of the bacterial community was assessed by t-RFLP and pyrosequencing analysis. The results showed that EMF treatment affected both planktonic and sessile community composition. EMFs were responsible for a shift in classes of Proteobacteria during development of the biofilm. It may be speculated that the EMF treatment affected particle solubility and/or microorganism hydration. This study indicated that EMFs modulated biofilm formation in natural water.

Surfactin from Bacillus subtilis displays an unexpected anti-Legionella activity.

A contaminant bacterial strain was found to exhibit an antagonistic activity against Legionella pneumophila, the causative agent of Legionnaires' disease. The bacterial strain was identified as a Bacillus subtilis and named B. subtilis AM1. PCR analysis revealed the presence of the sfp gene, involved in the biosynthesis of surfactin, a lipopeptide with versatile bioactive properties. The bioactive substances were extracted from AM1 cell-free supernatant with ethyl acetate and purified using reversed phase HPLC (RP-HPLC). Subsequent ESI-MS analyses indicated the presence of two active substances with protonated molecular ions at m/z 1008 and 1036 Da, corresponding to surfactin isoforms. Structures of lipopeptides were further determined by tandem mass spectrometry and compared to the spectra of a commercially available surfactin mixture. Surfactin displays an antibacterial spectrum almost restricted to the Legionella genus (MICs range 1-4 µg/mL) and also exhibits a weak activity toward the amoeba Acanthamoeba castellanii, known to be the natural reservoir of L. pneumophila. Anti-biofilm assays demonstrated that 66 µg/mL of surfactin successfully eliminated 90 % of a 6-day-old biofilm. In conclusion, this study reveals for the first time the potent activity of surfactin against Legionella sp. and preformed biofilms thus providing new directions toward the use and the development of lipopeptides for the control of Legionella spread in the environment.

Insights into betulinic acid as a promising molecule to fight the interkingdom biofilm Staphylococcus aureus-Candida albicans.

The demand for antibiofilm molecules has increased over several years due to their potential to fight biofilm-associated infections, such as those including the interkingdom Staphylococcus aureus-Candida albicans occurring in clinical settings worldwide. Recently, we identified a pentacyclic triterpenoid compound, betulinic acid, from invasive macrophytes, with interesting antibiofilm properties. The aim of the present study was to provide insights into the mechanism of action of betulinic acid against the clinically relevant bi-species S. aureus-C. albicans biofilms. Microscopy examinations, flow cytometry and crystal violet assays confirmed that betulinic acid was effective at damaging mature S. aureus-C. albicans biofilms or inhibiting their formation, reducing biofilm biomass by 70% on average and without microbicidal activity. The results suggested an action of betulinic acid on cell membranes, inducing changes in properties such as composition, hydrophobicity and fluidity as observed in C. albicans, which may hinder the early adhesion step, biofilm growth and the physical interactions of both microbial species. Further results of real-time polymerase chain reaction argued in favour of a reduction in S. aureus-C. albicans physical interaction due to betulinic acid by the modulation of biofilm-related gene expression, as observed in early stages of biofilm formation. This study revealed the potential of betulinic acid as a candidate agent for the prevention and treatment of S. aureus-C. albicans biofilm-related infections.

Sensitivity of Legionella pneumophila to phthalates and their substitutes.

Phthalates constitute a family of anthropogenic chemicals developed to be used in the manufacture of plastics, solvents, and personal care products. Their dispersion and accumulation in many environments can occur at all stages of their use (from synthesis to recycling). However, many phthalates together with other accumulated engineered chemicals have been shown to interfere with hormone activities. These compounds are also in close contact with microorganisms that are free-living, in biofilms or in microbiota, within multicellular organisms. Herein, the activity of several phthalates and their substitutes were investigated on the opportunistic pathogen Legionella pneumophila, an aquatic microbe that can infect humans. Beside showing the toxicity of some phthalates, data suggested that Acetyl tributyl citrate (ATBC) and DBP (Di-n-butyl phthalate) at environmental doses (i.e. 10-6 M and 10-8 M) can modulate Legionella behavior in terms of motility, biofilm formation and response to antibiotics. A dose of 10-6 M mostly induced adverse effects for the bacteria, in contrast to a dose of 10-8 M. No perturbation of virulence towards Acanthamoeba castellanii was recorded. These behavioral alterations suggest that L. pneumophila is able to sense ATBC and DBP, in a cross-talk that either mimics the response to a native ligand, or dysregulates its physiology.

Legionella spp. All Ears? The Broad Occurrence of Quorum Sensing Elements outside Legionella pneumophila.

Legionella spp. are ubiquitous bacteria principally found in water networks and ∼20 species are implicated in Legionnaire's disease. Among them, Legionella pneumophila is an intracellular pathogen of environmental protozoa, responsible for ∼90% of cases in the world. Legionella pneumophila regulates in part its virulence by a quorum sensing system named "Legionella quorum sensing," composed of a signal synthase LqsA, two histidine kinase membrane receptors LqsS and LqsT and a cytoplasmic receptor LqsR. To date, this communication system was only found in L. pneumophila. Here, we investigated 58 Legionella genomes to determine the presence of a lqs cluster or homologous receptors using TBlastN. This analysis revealed three categories of species: 19 harbored a complete lqs cluster, 20 did not possess lqsA but maintained the receptor lqsR and/or lqsS, and 19 did not have any of the lqs genes. No correlation was observed between pathogenicity and the presence of a quorum sensing system. We determined by RT-qPCR that the lqsA gene was expressed at least in four strains among different species available in our laboratory. Furthermore, we showed that the lqs genomic region was conserved even in species possessing only the receptors of the quorum sensing system, indicating an ancestral acquisition and various loss dynamics during evolution. This system could therefore function in interspecific communication as well.

Isopod holobionts as promising models for lignocellulose degradation.

BACKGROUND: Isopods have colonized all environments, partly thanks to their ability to decompose the organic matter. Their enzymatic repertoire, as well as the one of their associated microbiota, has contributed to their colonization success. Together, these holobionts have evolved several interesting life history traits to degrade the plant cell walls, mainly composed of lignocellulose. It has been shown that terrestrial isopods achieve lignocellulose degradation thanks to numerous and diverse CAZymes provided by both the host and its microbiota. Nevertheless, the strategies for lignocellulose degradation seem more diversified in isopods, in particular in aquatic species which are the least studied. Isopods could be an interesting source of valuable enzymes for biotechnological industries of biomass conversion. RESULTS: To provide new features on the lignocellulose degradation in isopod holobionts, shotgun sequencing of 36 metagenomes of digestive and non-digestive tissues was performed from several populations of four aquatic and terrestrial isopod species. Combined to the 15 metagenomes of an additional species from our previous study, as well as the host transcriptomes, this large dataset allowed us to identify the CAZymes in both the host and the associated microbial communities. Analyses revealed the dominance of Bacteroidetes and Proteobacteria in the five species, covering 36% and 56% of the total bacterial community, respectively. The identification of CAZymes and new enzymatic systems for lignocellulose degradation, such as PULs, cellulosomes and LPMOs, highlights the richness of the strategies used by the isopods and their associated microbiota. CONCLUSIONS: Altogether, our results show that the isopod holobionts are promising models to study lignocellulose degradation. These models can provide new enzymes and relevant lignocellulose-degrading bacteria strains for the biotechnological industries of biomass conversion.

The shutting down of the insulin pathway: a developmental window for Wolbachia load and feminization.

Using the isopod Armadillidium vulgare as a case study, we review the significance of the "bacterial dosage model", which connects the expression of the extended phenotype to the rise of the Wolbachia load. In isopods, the Insulin-like Androgenic Gland hormone (IAG) induces male differentiation: Wolbachia feminizes males through insulin resistance, presumably through defunct insulin receptors. This should prevent an autocrine development of the androgenic glands so that females differentiate instead: feminization should translate as IAG silencing and increased Wolbachia load in the same developmental window. In line with the autocrine model, uninfected males expressed IAG from the first larval stage on, long before the androgenic gland primordia begin to differentiate, and exponentially throughout development. In contrast in infected males, expression fully stopped at stage 4 (juvenile), when male differentiation begins. This co-occurred with the only significant rise in the Wolbachia load throughout the life-stages. Concurrently, the raw expression of the bacterial Secretion Systems co-increased, but they were not over-expressed relative to the number of bacteria. The isopod model leads to formulate the "bacterial dosage model" throughout extended phenotypes as the conjunction between bacterial load as the mode of action, timing of multiplication (pre/post-zygotic), and site of action (soma vs. germen).

Iron Availability Modulates the Persistence of Legionella pneumophila in Complex Biofilms.

Legionella pneumophila is a pathogenic bacteria found in biofilms in freshwater. Iron is an essential nutrient for L. pneumophila growth. In this study, complex biofilms were developed using river water spiked with L. pneumophila, and the persistence of L. pneumophila in these complex biofilms was evaluated. In order to study the role of iron in the persistence of L. pneumophila, river water was supplied with either iron pyrophosphate or iron chelators (deferoxamine mesylate, DFX for ferric iron and dipyridyl, DIP for ferrous iron) to modulate iron availability. The addition of iron pyrophosphate and DFX did not markedly affect the persistence of L. pneumophila in the biofilms, whereas that of DIP had a beneficial effect. Since DIP specifically chelates ferrous iron, we hypothesized that DIP may protect L. pneumophila from the deleterious effects of ferrous iron. In conclusion, ferrous iron appears to be important for the persistence of L. pneumophila in complex biofilms. However, further studies are needed in order to obtain a better understanding of the role of ferrous iron in the behavior of this bacterium in the environment.

Efficacy of dental unit waterlines disinfectants on a polymicrobial biofilm.

Due to their high surface-volume ratio, their laminar flow and frequent stagnation periods, dental unit waterlines (DUWL) foster the attachment of microorganisms and the development of biofilm, resulting in the continuous contamination of the outlet water from dental units; this contamination may be responsible for a potential risk of infection due to the exposure of patients and medical staff to droplet inhalation or splashed water. In this study, the anti-biofilm activity of three disinfectants recommended by dental unit manufacturers -Calbenium(©), Oxygenal 6(©) and Sterispray(©) - was evaluated. A dynamic model simulating DUWL conditions was developed and polymicrobial biofilms containing bacteria (Pseudomonas aeruginosa), fungi (Candida albicans) and Free Living Amoeba (FLA: Vermamoeba vermiformis) were allowed to form. The ability of disinfectants to reduce biofilm formation or to eradicate an already formed biofilm was evaluated. Results showed the various effects of the tested disinfectants according to their composition, concentration and the targeted species. V. vermiformis was resistant to disinfectants, regardless of the tested concentrations and the concentrations recommended by manufacturers were not the most appropriate. Results also showed that Calbenium(©) was the most effective disinfectant to reduce already formed biofilms; its maximum efficiency was observed from 0.5% on both P. aeruginosa and C. albicans compared to 2 and 3% respectively for Sterispray(©). The maximum efficiency of Oxygenal(©) was observed from 3% on P. aeruginosa but Oxygenal(©) was unable to totally eliminate C. albicans in the tested conditions, contrary to other disinfectants. Calbenium(©) was able to prevent biofilm formation efficiently even if it displayed no prophylactic activity against V. vermiformis. Overall, the FLA survival may contribute to maintaining other species. Finally the tested disinfectants were partially active against sessile microorganisms and more suitable concentrations could be used to increase their efficacy. Their use in a prophylactic rather than curative way should be recommended.

Wolbachia infect ovaries in the course of their maturation: last minute passengers and priority travellers?

Wolbachia are widespread endosymbiotic bacteria of arthropods and nematodes. Studies on such models suggest that Wolbachia's remarkable aptitude to infect offspring may rely on a re-infection of ovaries from somatic tissues instead of direct cellular segregation between oogonia and oocytes. In the terrestrial isopod Armadillidium vulgare, Wolbachia are vertically transmitted to the host offspring, even though ovary cells are cyclically renewed. Using Fluorescence in situ hybridization (FISH), we showed that the proportion of infected oocytes increased in the course of ovary and oocyte maturation, starting with 31.5% of infected oocytes only. At the end of ovary maturation, this proportion reached 87.6% for the most mature oocytes, which is close to the known transmission rate to offspring. This enrichment can be explained by a secondary acquisition of the bacteria by oocytes (Wolbachia can be seen as last minute passengers) and/or by a preferential selection of oocytes infected with Wolbachia (as priority travellers).

Intra-amoeba multiplication induces chemotaxis and biofilm colonization and formation for Legionella.

Legionella pneumophila, a facultative intracellular bacterium, is the causative agent of legionellosis. In the environment this pathogenic bacterium colonizes the biofilms as well as amoebae, which provide a rich environment for the replication of Legionella. When seeded on pre-formed biofilms, L. pneumophila was able to establish and survive and was only found at the surface of the biofilms. Different phenotypes were observed when the L. pneumophila, used to implement pre-formed biofilms or to form mono-species biofilms, were cultivated in a laboratory culture broth or had grown intracellulary within the amoeba. Indeed, the bacteria, which developed within the amoeba, formed clusters when deposited on a solid surface. Moreover, our results demonstrate that multiplication inside the amoeba increased the capacity of L. pneumophila to produce polysaccharides and therefore enhanced its capacity to establish biofilms. Finally, it was shown that the clusters formed by L. pneumophila were probably related to the secretion of a chemotaxis molecular agent.

Cannibalism and predation as paths for horizontal passage of Wolbachia between terrestrial isopods.

The alpha-proteobacteria Wolbachia are the most widespread endosymbionts in arthropods and nematodes. Mainly maternally inherited, these so-called sex parasites have selected several strategies that increase their vertical dispersion in host populations. However, the lack of congruence between the Wolbachia and their host phylogenies suggests frequent horizontal transfers. One way that could be used for horizontal Wolbachia transfers between individuals is predation. The aim of this study was to test whether horizontal passage of Wolbachia is possible when an uninfected terrestrial isopod eats an infected one. After having eaten Armadillidium vulgare harbouring Wolbachia, the predator-recipients (the two woodlice A. vulgare and Porcellio dilatatus dilatatus) that were initially Wolbachia-free were tested positive for the presence of Wolbachia both by quantitative PCR and Fluorescence in situ Hybridization (FISH). Even if the titers were low compared to vertically infected individuals, this constitutes the first demonstration of Wolbachia occurrence in various organs of an initially uninfected host after eating an infected one.

The immune cellular effectors of terrestrial isopod Armadillidium vulgare: meeting with their invaders, Wolbachia.

BACKGROUND: Most of crustacean immune responses are well described for the aquatic forms whereas almost nothing is known for the isopods that evolved a terrestrial lifestyle. The latter are also infected at a high prevalence with Wolbachia, an endosymbiotic bacterium which affects the host immune system, possibly to improve its transmission. In contrast with insect models, the isopod Armadillidium vulgare is known to harbor Wolbachia inside the haemocytes. METHODOLOGY/PRINCIPAL FINDINGS: In A. vulgare we characterized three haemocyte types (TEM, flow cytometry): the hyaline and semi-granular haemocytes were phagocytes, while semi-granular and granular haemocytes performed encapsulation. They were produced in the haematopoietic organs, from central stem cells, maturing as they moved toward the edge (TEM). In infected individuals, live Wolbachia (FISH) colonized 38% of the haemocytes but with low, variable densities (6.45±0.46 Wolbachia on average). So far they were not found in hyaline haemocytes (TEM). The haematopoietic organs contained 7.6±0.7×10(3)Wolbachia, both in stem cells and differentiating cells (FISH). While infected and uninfected one-year-old individuals had the same haemocyte density, in infected animals the proportion of granular haemocytes in particular decreased by one third (flow cytometry, Pearson's test = 12 822.98, df = 2, p<0.001). CONCLUSIONS/SIGNIFICANCE: The characteristics of the isopod immune system fell within the range of those known from aquatic crustaceans. The colonization of the haemocytes by Wolbachia seemed to stand from the haematopoietic organs, which may act as a reservoir to discharge Wolbachia in the haemolymph, a known route for horizontal transfer. Wolbachia infection did not affect the haemocyte density, but the quantity of granular haemocytes decreased by one third. This may account for the reduced prophenoloxidase activity observed previously in these animals.

Reproductive fitness and dietary choice behavior of the genetic model organism Caenorhabditis elegans under semi-natural conditions.

Laboratory breeding conditions of the model organism C. elegans do not correspond with the conditions in its natural soil habitat. To assess the consequences of the differences in environmental conditions, the effects of air composition, medium and bacterial food on reproductive fitness and/or dietary-choice behavior of C. elegans were investigated. The reproductive fitness of C. elegans was maximal under oxygen deficiency and not influenced by a high fractional share of carbon dioxide. In media approximating natural soil structure, reproductive fitness was much lower than in standard laboratory media. In seminatural media, the reproductive fitness of C. elegans was low with the standard laboratory food bacterium E. coli (γ-Proteobacteria), but significantly higher with C. arvensicola (Bacteroidetes) and B. tropica (ß-Proteobacteria) as food. Dietary-choice experiments in semi-natural media revealed a low preference of C. elegans for E. coli but significantly higher preferences for C. arvensicola and B. tropica (among other bacteria). Dietary-choice experiments under quasi-natural conditions, which were feasible by fluorescence in situ hybridization (FISH) of bacteria, showed a high preference of C. elegans for Cytophaga-Flexibacter-Bacteroides, Firmicutes, and ß-Proteobacteria, but a low preference for γ-Proteobacteria. The results show that data on C. elegans under standard laboratory conditions have to be carefully interpreted with respect to their biological significance.

Soil amoebae rapidly change bacterial community composition in the rhizosphere of Arabidopsis thaliana.

We constructed an experimental model system to study the effects of grazing by a common soil amoeba, Acanthamoeba castellanii, on the composition of bacterial communities in the rhizosphere of Arabidopsis thaliana. Amoebae showed distinct grazing preferences for specific bacterial taxa, which were rapidly replaced by grazing tolerant taxa in a highly reproducible way. The relative proportion of active bacteria increased although bacterial abundance was strongly decreased by amoebae. Specific bacterial taxa had disappeared already two days after inoculation of amoebae. The decrease in numbers was most pronounced in Betaproteobacteria and Firmicutes. In contrast, Actinobacteria, Nitrospira, Verrucomicrobia and Planctomycetes increased. Although other groups, such as betaproteobacterial ammonia oxidizers and Gammaproteobacteria did not change in abundance, denaturing gradient gel electrophoresis with specific primers for pseudomonads (Gammaproteobacteria) revealed both specific changes in community composition as well as shifts in functional genes (gacA) involved in bacterial defence responses. The resulting positive feedback on plant growth in the amoeba treatment confirms that bacterial grazers play a dominant role in structuring bacteria-plant interactions. This is the first detailed study documenting how rapidly protozoan grazers induce shifts in rhizosphere bacterial community composition.

Occurrence and diversity of bacterial communities in Tuber magnatum during truffle maturation.

Tuber magnatum, an ascomycetous fungus and obligate ectomycorrhizal symbiont, forms hypogeous fruit bodies, commonly called Italian white truffles. The diversity of bacterial communities associated with T. magnatum truffles was investigated using culture-independent and -dependent 16S rRNA gene-based approaches. Eighteen truffles were classified in three groups, representing different degrees of ascocarp maturation, based on the percentage of asci containing mature spores. The culturable bacterial fraction was (4.17 +/- 1.61) x 10(7), (2.60 +/- 1.22) x 10(7) and (1.86 +/- 1.32) x 10(6) cfu g(-1) for immature, intermediate and mature ascocarps respectively. The total of bacteria count was two orders of magnitude higher than the cfu g(-1) count. Sequencing results from the clone library showed a significant presence of alpha-Proteobacteria (634 of the 771 total clones screened, c. 82%) affiliated with Sinorhizobium, Rhizobium and Bradyrhizobium spp. The bacterial culturable fraction was generally represented by gamma-Proteobacteria (210 of the 384 total strains isolated, c. 55%), which were mostly fluorescent pseudomonads. Fluorescent in situ hybridization confirmed that alpha-Proteobacteria (85.8%) were the predominant components of truffle bacterial communities with beta-Proteobacteria (1.5%), gamma-Proteobacteria (1.9%), Bacteroidetes (2.1%), Firmicutes (2.4%) and Actinobacteria (3%) only poorly represented. Molecular approaches made it possible to identify alpha-Proteobacteria as major constituents of a bacterial component associated with T. magnatum ascoma, independently from the degree of maturation.