Phylogenetic Characterization and In Situ Detection of a Cytophaga-Flexibacter-Bacteroides Phylogroup Bacterium in Tuber borchii Vittad. Ectomycorrhizal Mycelium

Mycorrhizal ascomycetous fungi are obligate ectosymbionts that colonize the roots of gymnosperms and angiosperms. In this paper we describe a straightforward approach in which a combination of morphological and molecular methods was used to survey the presence of potentially endo- and epiphytic bacteria associated with the ascomycetous ectomycorrhizal fungus Tuber borchii Vittad. Universal eubacterial primers specific for the 5′ and 3′ ends of the 16S rRNA gene (16S rDNA) were used for PCR amplification, direct sequencing, and phylogenetic analyses. The 16S rDNA was amplified directly from four pure cultures of T. borchii Vittad. mycelium. A nearly full-length sequence of the gene coding for the prokaryotic small-subunit rRNA was obtained from each T. borchii mycelium studied. The 16S rDNA sequences were almost identical (98 to 99% similarity), and phylogenetic analysis placed them in a single unique rRNA branch belonging to the Cytophaga-Flexibacter-Bacteroides (CFB) phylogroup which had not been described previously. In situ detection of the CFB bacterium in the hyphal tissue of the fungus T. borchii was carried out by using 16S rRNA-targeted oligonucleotide probes for the eubacterial domain and the Cytophaga-Flexibacter phylum, as well as a probe specifically designed for the detection of this mycelium-associated bacterium. Fluorescent in situ hybridization showed that all three of the probes used bound to the mycelium tissue. This study provides the first direct visual evidence of a not-yet-cultured CFB bacterium associated with a mycorrhizal fungus of the genus Tuber.     

Competitive PCR for quantitation of a Cytophaga-Flexibacter-Bacteroides phylum bacterium associated with the Tuber borchii Vittad. mycelium

An uncultured bacterium associated with the ectomycorrhizal fungus Tuber borchii Vittad. was identified as a novel member of the Cytophaga-Flexibacter-Bacteroides group. Utilizing a quantitative PCR targeting the 16S rRNA gene, we relatively quantified this bacterium in the host. The estimated number of bacteria was found to be approximately 10(6) cells per 30-day-old T. borchii mycelium culture. This represents the first molecular attempt to enumerate an uncultured bacterium associated with a mycorrhizal fungus.     

New evidence for bacterial diversity in the ascoma of the ectomycorrhizal fungus Tuber borchii Vittad

The microbial community associated with ascocarps of the ectomycorrhizal fungus Tuber borchii Vittad. was studied by both cultivation and direct extraction of bacterial 16S rRNA gene (rDNA) sequence approaches. The inner part of six T. borchii ascoma collected in North-Central Italy was used to establish a bacterial culture collection and to extract the total genomic DNA to obtain a library of 16S rDNAs representative of the truffle bacterial community. Most of the isolates were affiliated to the gamma-Proteobacteria, mainly Fluorescent pseudomonads; some isolates were members of the Bacteroidetes group and Gram-positive bacteria, mostly Bacillaceae. The majority of the clones from the library were alpha-Proteobacteria showing significant similarity values, of greater than 97%, with members of the Sinorhizobium/Ensifer Group, Rhizobium and Bradyrhizobium spp. not previously identified as Tuber-associated bacteria. Only a few bacterial strains belonging to this bacterial subclass were found in the culture collection and isolated on a medium specific for Rhizobium-like organisms. A few clones were members of the beta- and gamma-Proteobacteria; as well as low and high G+C Gram-positive bacteria. Our findings clearly indicate that a dual approach increases the information obtained on the structural composition of a truffle bacterial community as compared to that derived via cultivation or direct recovery of 16S rDNA sequences alone.     

Molecular markers for the identification of the ectomycorrhizal fungus Tuber borchii

Five species of white truffle were classified using PCR-based techniques. RAPD (random amplified polymorphic DNA) fingerprints and specific pairs of primers were used. A RAPD fragment was constant in Tuber borchii Vittad. isolates and polymorphic among the other species. Two molecular markers specific for T. borchii were developed from the sequence of the non-polymorphic RAPD fragment and from regions flanking the 5'-3' ends of a truffle gene. These markers were applied in the identification of T. borchii fruit bodies, mycelia and mycorrhizas, allowing us to monitor the development of this fungus during its entire life cycle.     

Three different forms of hexokinase are identified during Tuber Borchii mycelium growth

Truffles are ectomycorrhizal fungi which have a great dependence on carbohydrates supplied by their host plants. The catabolism of hexoses in the mycobiont is important for the production of energy, and the first enzyme in the hexose assimilation pathways is hexokinase. This study reports differences in the expression of this enzyme during the growth of Tuber borchii Vittad. mycelium (strain ATCC 96540). Three hexokinase activities (HKM1, HKM2 and HKM3) were isolated by anion-exchange chromatography and partially purified. HKM1 and HKM2 were present in the linear phase at 15-50 days of growth. Two remarkable differences were found in the sugar-phosphorylating activity and stability of HKM1 and HKM2. HKM2 did not phosphorylate the fructose and it was present in the chromatographic profile only when substrates such as glucose, glucosamine or mannose were added to the extraction buffer. On the contrary, HKM1 utilized also fructose and was detected under all the experimental conditions used. HKM3 was the only molecular form observed after 70 days, when the fungus growth had reached a plateau. To our knowledge these results represent the first evidence for the presence in T. borchii mycelium of three distinct enzymatic forms of hexokinase which are differently expressed during growth of the fungus.     

Adhesion to hyphal matrix and antifungal activity of Pseudomonas strains isolated from Tuber borchii ascocarps

Pseudomonas spp. isolates from Tuber borchii ascocarps, known to be able to produce phytoregulatory and biocontrol substances in pure culture, were used to perform studies on their possible physiological role in nature. Antimycotic activity was confirmed against fungal contaminants isolated from the ascocarps, suggesting that populations associated with Tuber borchii fruit bodies may play a role in the maintenance of ascocarp health. Fifty-five percent of strains tested were also able to release metabolites which affected T. borchii mycelial growth and morphogenesis in culture. On the contrary, growth of the arbuscular mycorrhizal fungus Glomus mosseae and the ectomycorrhizal fungus Laccaria bicolor, putative competitors of Tuber for mycorrhizal infection sites on roots, was not influenced by the presence of any bacterial strain. The possibility that these bacteria, which show antifungal activity and fungal growth modulation activities, might be incorporated in the developing ascocarp by means of their preferential adhesion to Tuber mycelium is discussed.     

Cloning, expression, and characterization of the hxk-1 Gene from the white truffle Tuber borchii vittad.: A first step toward understanding sugar metabolism.

Recent biochemical investigations of Tuber borchii Vittad. mycelium have demonstrated the presence of three distinct forms of hexokinase (HK(M1), HK(M2), and HKM3). In the investigation described here, a gene coding for hexokinase (hxk-1) from T. borchii was isolated and characterized. The hxk-1 gene is characterized by an ORF of 1494 nucleotides and codes for a polypeptide of 497 aa. The gene was overexpressed in Escherichia coli, and the recombinant protein was kinetically characterized. The K(cat) value for fructose is in agreement with the data reported for the hexokinase of Yarrowia lipolytica, the Km for ATP is not dependent on the sugar used, and the enzyme is not inhibited by trehalose 6-phosphate or glucose 6-phosphate. The biochemical characteristics confirm that this enzyme is a hexokinase, as suggested by the Pileup results, and it corresponds to the HKM1 isoform. This work represents the first characterization of the key enzyme of the glycolytic pathway and the related gene in a Tuber species.     

Expression and purification of a Tuber borchii fruitbody-specific protein, TBF-1, from Escherichia coli: generation of polyclonal antibodies

TBF-1 is a fruitbody-specific protein present in the white truffle species Tuber borchii Vittad. A similar protein has been found only in the closely related species Tuber dryophilum (TDF-1), but not in other truffles. The protein from T. borchii was overexpressed as fusion protein in E. coli and was purified to homogeneity by affinity chromatography. Recombinant protein was used for generating polyclonal antibodies. The antiserum strongly reacted with TBF-1, weakly recognized TDF-1, and did not detect correlate band in the other white truffle species. The high level of expression of this protein in the fruitbody and the specificity of the antibody anti-TBF-1 make it possible to set up a diagnostic tool for detecting these species in natural samples and foodstuffs.     

Molecular characterisation of a Tuber borchii Smt3 gene.

Tbsmt3 gene from the ectomychorrizal fungus Tuber borchii was identified and sequenced. The Tbsmt3 gene encodes for a protein sharing significant amino acid homology with the yeast SMT3, a ubiquitin-like protein that is post-translationally attached to several proteins involved in many cellular processes. The comparison between the Tbsmt3 genomic and cDNA sequences established that the encoding sequence is interrupted by an intron of 312 bp. Southern blot analysis revealed only one copy of Tbsmt3 gene in the T. borchii genome. Tbsmt3 is expressed in all phases of T. borchii life cycle: mycelium, ectomycorrhiza and ascoma. However, the Tbsmt3 mRNA decreased during fruit body maturation.     

Members of the Cytophaga–Flavobacterium–Bacteroides phylum as intracellular bacteria of acanthamoebae: proposal of 'Candidatus Amoebophilus asiaticus'

Three Gram-negative, rod-shaped bacteria that were found intracellularly in two environmental and one clinical Acanthamoeba sp. isolates were analysed. Two endocytobionts showing a parasitic behaviour were propagated successfully outside their amoebal host cells and were identified subsequently by comparative 16S rRNA sequence analysis as being most closely affiliated with Flavobacterium succinicans (99% 16S rRNA sequence similarity) or Flavobacterium johnsoniae (98% 16S rRNA sequence similarity). One endocytobiont could neither be cultivated outside its original Acanthamoeba host ( Acanthamoeba sp. TUMSJ-321) nor transferred into other amoebae. Electron microscopy revealed that the amoebal trophozoites and cysts were almost completely filled with cells of this endosymbiont which are surrounded by a host-derived membrane. According to 16S rRNA sequence analysis, this endosymbiont could also be assigned to the Cytophaga – Flavobacterium – Bacteroides (CFB) phylum, but was not closely affiliated to any recognized species within this phylogenetic group (less than 82% 16S rRNA sequence similarity). Identity and intracellular localization of this endosymbiont were confirmed by application of a specific fluorescently labelled 16S rRNA-targeted probe. Based on these findings, we propose classification of this obligate Acanthamoeba endosymbiont as ' Candidatus Amoebophilus asiaticus'. Comparative 18S rRNA sequence analysis of the host of ' Candidatus Amoebophilus asiaticus' revealed its membership with Acanthamoeba 18S rDNA sequence type T4 that comprises the majority of all Acanthamoeba isolates.     

Tests of species concepts of the small,white, European group of <Emphasis Type="Italic">Tuber</Emphasis> spp. based on morphology and rDNA ITS sequences with special reference to <Emphasis Type="Italic">Tuber rapaeodorum</Emphasis>

Several taxonomic problems arise in the group of small, white European truffles, probably due to the over-emphasized significance of certain morphological features of ascomata. The distinction between Tuber rapaeodorum Tul. & C. Tul. and Tuber borchii Vittad. and Tuber puberulum Berk. & Broome has not been accepted in several recent studies. Furthermore, the existence of T. rapaeodorum been questioned in some recent synopses of the genus. We conducted microscopic and ITS sequence investigations of 31 herbarium specimens. Using morphological features such as peridium structure, form and size of spores and dermatocystidia and spore numbers per ascus, we could distinguish T. borchii, T. foetidum Vittad., T. maculatum Vittad., Tuber puberulum, and T. rapaeodorum. Analysis of whole ITS sequences showed sharp differences among the morphologically separated groups. Neighbour-joining and parsimony methods produced highly supported branches and confirmed the identity of these species.     

Dual staining of natural bacterioplankton with 4',6-diamidino-2-phenylindole and fluorescent oligonucleotide probes targeting kingdom-level 16S rRNA sequences.

A method for quantifying eubacterial cell densities in dilute communities of small bacterioplankton is presented. Cells in water samples were stained with 4',6-diamidino-2-phenylindole (DAPI), transferred to gelatin-coated slides, and hybridized with rhodamine-labeled oligonucleotide probes specific for kingdom-level 16S rRNA sequences. Between 48 and 69% of the cells captured on membrane filters were transferred to gelatin-coated slides. The number of DAPI-stained cells that were visualized with eubacterial probes varied from 35 to 67%. Only 2 to 4% of these cells also fluoresced following hybridization with a probe designed to target a eukaryotic 16S rRNA sequence. Between 0.1 and 6% of the bacterioplankton in these samples were autofluorescent and may have been mistaken as cells that hybridized with fluorescent oligonucleotide probes. Dual staining allows precise estimates of the efficiency of transfers of cells to gelatin films and can be used to measure the percentage of the total bacterioplankton that also hybridize with fluorescent oligonucleotide probes, indicating specific phylogenetic groups.     

Broad range DNA probes for detecting and amplifying eubacterial nucleic acids

In this report we describe and characterize two oligomer probes that are broadly homologous to conserved eubacterial 16S ribosomal RNA (rRNA) sequences not present in human 18 rRNA or human mitochondrial 12S rRNA. One or both of the probes can detect all of 23 phylogenetically diverse eubacterial nucleic acids against which they were tested by dot blot hybridization. A sensitivity of about 1 bacterium per 10 eukaryotic cells was achieved. By using these oligomer sequences or their complements as primers in the polymerase chain reaction (PCR), the equivalent of 1 pg of E. coli DNA was detected in the presence of a large excess of eukaryotic DNA. Information useful for partial phylogenetic classification of detected organisms may be obtained by direct sequence analysis of the amplified DNA and comparison with known sequences or catalogs. Such broadly homologous probes offer advantages over more narrowly specific probes for detecting organisms whose identity is unknown. They could thus be employed for recognizing infection by organisms that cannot be cultured as may occur, for example, in tissue culture or in plant or animal diseases of unknown cause, provided the probes fail to hybridize with host nucleic acids.     

Chitin synthase homologs in three ectomycorrhizal truffles

Abstract Degenerate PCR primers were used to amplify a conserved gene portion coding chitin synthase from genomic DNA of six species of ectomycorrhizal truffles. DNA was extracted from both hypogeous fruitbodies and in vitro growing mycelium of Tuber borchii . A single fragment of about 600 bp was amplified for each species. The amplification products from Tuber magnatum, T. borchii and T. ferrugineum were cloned and sequenced, revealing a high degree of identity (91.5%) at the nucleotide level. On the basis of the deduced amino acid sequences these clones were assigned to class II chitin synthase. Southern blot experiments performed on genomic DNA showed that the amplification products derive from a single copy gene. Phylogenetic analysis of the nucleotide sequences of class II chitin synthase genes confirmed the current taxonomic position of the genus Tuber , and suggested a close relationship between T. magnatum and T. uncinatum .     

The Tuber borchii fruiting body-specific protein TBF-1, a novel lectin which interacts with associated Rhizobium species

Lectins, proteins that are able to bind carbohydrate structures, are typically involved in cell recognition mechanisms. We demonstrate here that TBF-1, the main soluble protein in the Tuber borchii Vittad. fruiting body, is a phase-specific lectin that is able selectively to bind the exopolysaccharides produced by ascoma-associated Rhizobium spp. Characterization of TBF-1 was performed using both the protein purified from the truffles and the recombinant protein overexpressed in Escherichia coli. The two proteins exhibit the same hemagglutination activity toward rabbit red blood cells and the same sugar binding specificity. The discovery of lectin activity for TBF-1 led us to propose revising the protein name to 'T. borchii fruiting body lectin 1' with the acronym TBFL-1.     

Dual staining of natural bacterioplankton with 4',6-diamidino-2-phenylindole and fluorescent oligonucleotide probes targeting kingdom-level 16S rRNA sequences.

A method for quantifying eubacterial cell densities in dilute communities of small bacterioplankton is presented. Cells in water samples were stained with 4',6-diamidino-2-phenylindole (DAPI), transferred to gelatin-coated slides, and hybridized with rhodamine-labeled oligonucleotide probes specific for kingdom-level 16S rRNA sequences. Between 48 and 69% of the cells captured on membrane filters were transferred to gelatin-coated slides. The number of DAPI-stained cells that were visualized with eubacterial probes varied from 35 to 67%. Only 2 to 4% of these cells also fluoresced following hybridization with a probe designed to target a eukaryotic 16S rRNA sequence. Between 0.1 and 6% of the bacterioplankton in these samples were autofluorescent and may have been mistaken as cells that hybridized with fluorescent oligonucleotide probes. Dual staining allows precise estimates of the efficiency of transfers of cells to gelatin films and can be used to measure the percentage of the total bacterioplankton that also hybridize with fluorescent oligonucleotide probes, indicating specific phylogenetic groups.     

Structural analysis of the rDNA intergenic spacer of Tuber borchii

The sequence and characterisation of the entire nuclear rDNA intergenic spacer (IGS) for the genus Tuber are presented. Sequence analyses showed that the organisation of the Tuber borchii rDNA IGS is typical of rDNA spacers, consisting of a central repetitive region and flanking unique sequences on either side. Direct repeats, symmetry elements, tandem repeats and possible areas of recombination were found. The putative ends of the 25S and 17S rDNA were identified. The presence of 5S rDNA in the IGS region was excluded.     

一株拮抗姜瘟青枯假单胞杆菌的链霉菌的分离及鉴定

从生姜田土中分离到一株对姜瘟青枯假单胞杆菌(PseudomonassolanacarumSmith)有强拮抗作用的链霉菌菌株SR 11,研究拮抗性表明,对革兰氏阳性细菌、革兰氏阴性细菌以及多种病原真菌均有很强的抑制作用。对该菌株进行形态特征、培养特征、生理生化、细胞壁组分分析及16SrDNA序列分析。基内菌丝无横隔、不断裂,气生菌丝多分枝;孢子丝波曲至螺旋形,孢子椭圆形,表面光滑。细胞壁化学组分Ⅰ型,糖型C。在培养成熟后,气丝变为灰色,可闻到浓烈的土味。以16SrDNA序列为基础构建了包括13株相关种属细菌在内的系统发育树,其中,与12个模式链霉菌株的16SrDNA序列的同源性为96 5 %～98 3%。     

MOLECULAR IDENTIFICATION OF A BACTERIUM ASSOCIATED WITH GALL FORMATION IN THE MARINE RED ALGA PRIONITIS LANCEOLATA1

The polymerase chain reaction (PCR) was used to amplify eubacterial small-subunit (16S) ribosomal DNA (rDNA) genes from galls of the marine red alga Prionitis lanceolata Harvey (Gigartinales). These tumors consist of hypertrophied algal cells containing large numbers of intercellular bacteria that remain uncultivable. PCR-amplified 16S rDNAs from surface-sterilized gall tissue plugs were cloned, sequenced, and analyzed by alignment to available small-subunit rRNA sequences (University of Illinois Ribosomal Database Project). Variable regions were identified and used to construct a fluorescently labeled, species-specific oligodeoxynucleotide probe for whole cell in situ hybridization to the gall symbiont. Probe 949 (PLANC.949) localized the P. lanceolata bacterial symbiont in preparations from mature gall tissue. This probe did not hybridize to the rDNA of closely related bacteria included as controls in the same hybridization reactions, In situ hybridization revealed the presence of the same bacterium in association with P. lanceolata gall formation from three central California localities. Distance and parsimony analyses suggest that this organism is a member of the Proteobacteria (alpha subdivision; Rhodobacter group) and is most closely related to Roseobacter denitrificans .