Publications

4368

ABSTRACT The characterization of transcriptional start sites of 14 genes encoded by the extremely AT-rich genome of “ Candidatus Blochmannia floridanus” revealed a high degree of conservation with the RpoD promoter consensus sequence of the free-living relative Escherichia coli . Moreover, in agreement with the presence of the alternative heat shock sigma factor RpoH in “ Ca . Blochmannia,” typical RpoH-dependent promoters were identified. However, no heat shock response resembling that of E. coli could be detected in “ Ca . Blochmannia.”

In North America, elm yellows, aster yellows (AY), and X-disease phytoplasmas have been detected in American grapevines (1), and recently, Bois noir was detected in Canadian vineyards from British Columbia (BC) and Ontario (ON) (2). Typical symptoms of grapevine yellows (GY) include leaf rolling and chlorosis, uneven or total lack of lignification of canes, flower abortion or berry withering, and stunting. In 2006 and 2007, independent surveys were conducted by the Canadian Food Inspection Agency (CFIA) and Agriculture and Agri-Food Canada (AAFC) to detect phytoplasmas in Canadian vineyards containing different cultivars in BC, ON, Québec (QC), Nova Scotia, New Brunswick, and Prince Edward Island. The CFIA collected and tested 651 fresh leaf samples from recently imported grapevines and older grapevines in the same or neighboring blocks displaying symptoms typical of those associated with disease caused by phytoplasmas. Many vineyards were surveyed only once. AAFC collected and tested 3,485 samples from symptomatic and asymptomatic grapevines from established vineyards in ON, BC, and QC. The same vineyards were sampled in ON and BC both years; QC vineyards were only sampled in 2007. AAFC-collected leaf samples were freeze dried and stored at –20°C before processing. CFIA samples were tested by a modified real-time PCR assay and TaqMan probe targeting the 16S ribosomal RNA gene that detects a wide range of known phytoplasmas (2). Positive samples were confirmed by conventional PCR using the phytoplasma-specific primers P1/P7 (3) and the resulting ~1,800-bp fragment was cloned and sequenced as previously described (2). DNA extracted by AAFC was amplified by nested PCR technology with universal phytoplasma specific primer pairs P1/P6 and R16R2/R16F2 (3) and the resulting 1,200-bp fragment was cloned and sequenced. Two plants, one located in ON in 2006 and the other in BC in 2007, were found to be infected with an AY-like phytoplasma by the CFIA. The phytoplasmas detected in both infected plants had a 99.9% nt sequence identity with AY phytoplasma sequences from GenBank (Accession Nos. AF222063 and AY665676, respectively), with the BC isolate also showing 100% identity to a strain of AY, ash witches'-broom phytoplasma (GenBank Accession No. AY566302). AAFC detected phytoplasma DNA in both years in a total of 17 symptomatic plants and 21 asymptomatic plants from different vine varieties in ON, BC, and QC. Positive samples were found to have a 99.0% nt sequence identity to AY subgroup 16SrI-A (GenBank Accession No. AY180956). Sequences were exchanged for confirmation of phytoplasma identity and were deposited in Genbank under Accession Nos. FJ659844 and FJ824597. Phytoplasma strains were identified for all plants in which phytoplasmas were detected. Results show that AY is present in vineyards in the provinces of ON, BC, and QC. To our knowledge, this is the first report of AY being detected in grapevines in Canada. References: (1) E. Boudon-Padieu. Bull. O I V, 79:299, 2003. (2) M. Rott et al. Plant Dis. 91:1682, 2007. (3) E. Tanne et al. Phytopathology 91:741, 2001.

Summary Multicellular magnetotactic prokaryotes (MMPs) represent highly organized, spherical and motile aggregates of 10–40 bacterial cells containing magnetosomes. Although consisting of different cells, each with its own magnetosomes and flagellation, MMPs orient themselves within a magnetic field and exhibit magnetotaxis. So far, MMPs have only been found in several North and South American coastal lagoons and salt marshes. In the present study, a novel type of MMP was discovered in coastal tidal sand flats of the North Sea. High‐resolution scanning electron microscopy revealed the presence of bullet‐shaped magnetosomes which were aligned in several parallel chains. Within each aggregate, the magnetosome chains of individual cells were oriented in the same direction. Energy dispersive X‐ray (EDX) analysis showed that the magnetosomes are composed of iron sulfide. This particular morphology and arrangement of magnetosomes has previously not been reported for other MMPs. 16S rRNA gene sequence analysis revealed a single phylotype which represented a novel phylogenetic lineage with ≥ 4% sequence divergence to all previously described MMP sequences and was related to the dissimilatory sulfate‐reducing Desulfosarcina variabilis within the family Desulfobacteraceae of the subphylum Deltaproteobacteria . Fluorescence in situ hybridization with a specific oligonucleotide probe revealed that all MMPs in the tidal flat sediments studied belonged to the novel phylotype. Within each MMP, all bacterial cells showed a hybridization signal, indicating that the aggregates are composed of cells of the same phylotype. Genes for dissimilatory sulfite reductase ( dsr AB) and dissimilatory adenosine‐5′‐phosphate reductase ( apr A) could be detected in purified MMP samples, suggesting that MMPs are capable of sulfate reduction. Chemotaxis assays with 41 different test compounds yielded strong responses towards acetate and propionate, whereas other organic acids, alcohols, sugars, sugar alcohols or sulfide did not elicit any response. By means of its coordinated magnetotaxis and chemotaxis, the novel type of MMP is well adapted to the steep chemical gradients which are characteristic for intertidal marine sediments.

Bamboos are ecologically and economically valuable plants. Young shoots of almost all species are edible, either raw or cooked, and are major components of Asian cuisine while culms are used for furniture or handicrafts as well as fuel wood. Symptoms indicative of phytoplasma disease were observed on Sasa fortunei (van Houtte) Fiori during a survey in Yangling, Shaanxi Province, China during 2007. Symptoms included internode shortening, a mosaic pattern on leaves of diminished size, stunted growth, and death of entire plants. Total nucleic acids were extracted from leaf veins of 10 diseased plants and 6 symptomless plants with a modified cetyltrimethylammoniumbromide (CTAB) method (1). Phytoplasma infection of plants was demonstrated by a nested PCR assay employing primer pair R16mF2/R16mR1 followed by R16F2n/R16R2 (2), which generated a 16S rDNA product of approximately 1.2 kb from all symptomatic plants only. Restriction fragment length polymorphism analysis by digestion of nested PCR products with restriction enzymes AluI and MseI indicated that S. fortunei plants contained group 16SrI (aster yellows), subgroup B phytoplasmas. Sequencing and phylogenetic analysis of phytoplasma 16S rDNA from S. fortunei (GenBank Accession No. FJ501958) revealed this strain to be very similar (99.7 and 99.6%, respectively) to phytoplasmas previously associated with Henon bamboo (Phyllostachys nigra) witches'-broom (GenBank Accession No. AB242433) (4) and sasa (S. borealis) witches'-broom (GenBank Accession No. AB293421) in Korea but less so (98.6%) to bamboo (Phyllostachys spp.) witches'-broom (GenBank Accession No. AY635145) disease in China (3). To our knowledge, this is the first report of a ‘Ca. Phytoplasma asteris’-related strain infecting S. fortunei in China. References: (1) E. Angelini et al. Vitis 40:79, 2001. (2) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:144, 1996. (3) C. Hong et al. Plant Prot. 31:39, 2005. (4) H. Jung et al. J. Gen. Plant Pathol. 72:261, 2006.

ABSTRACT Insect intestinal tracts harbor several novel, deep-rooting clades of as-yet-uncultivated bacteria whose biology is typically completely unknown. Here, we report the isolation of the first representative of the termite group 1 (TG1) phylum from sterile-filtered gut homogenates of a humivorous scarab beetle larva. Strain Pei191 T is a mesophilic, obligately anaerobic ultramicrobacterium with a gram-negative cell envelope. Cells are typically rod shaped, but cultures are pleomorphic in all growth phases (0.3 to 2.5 μm long and 0.17 to 0.3 μm wide). The isolate grows heterotrophically on sugars and ferments d -galactose, d -glucose, d -fructose, d -glucosamine, and N -acetyl- d -glucosamine to acetate, ethanol, hydrogen, and alanine as major products but only if amino acids are present in the medium. PCR-based screening and comparative 16S rRNA gene sequence analysis revealed that strain Pei191 T belongs to the “intestinal cluster,” a lineage of hitherto uncultivated bacteria present in arthropod and mammalian gut systems. It is only distantly related to the previously described so-called “endomicrobia” lineage, which comprises mainly uncultivated endosymbionts of termite gut flagellates. We propose the name “ Elusimicrobium minutum ” gen. nov., sp. nov. (type strain, Pei191 T = ATCC BAA-1559 T = JCM 14958 T ) for the first isolate of this deep-branching lineage and the name “ Elusimicrobia ” phyl. nov. for the former TG1 phylum.

Summary Many parasitic bacteria live in the cytoplasm of multicellular animals, but only a few are known to regularly invade their nuclei. In this study, we describe the novel bacterial parasite “ Candidatus Endonucleobacter bathymodioli” that invades the nuclei of deep‐sea bathymodiolin mussels from hydrothermal vents and cold seeps. Bathymodiolin mussels are well known for their symbiotic associations with sulfur‐ and methane‐oxidizing bacteria. In contrast, the parasitic bacteria of vent and seep animals have received little attention despite their potential importance for deep‐sea ecosystems. We first discovered the intranuclear parasite “ Ca. E. bathymodioli” in Bathymodiolus puteoserpentis from the Logatchev hydrothermal vent field on the Mid‐Atlantic Ridge. Using primers and probes specific to “ Ca . E. bathymodioli” we found this intranuclear parasite in at least six other bathymodiolin species from vents and seeps around the world. Fluorescence in situ hybridization and transmission electron microscopy analyses of the developmental cycle of “ Ca . E. bathymodioli” showed that the infection of a nucleus begins with a single rod‐shaped bacterium which grows to an unseptated filament of up to 20 μm length and then divides repeatedly until the nucleus is filled with up to 80 000 bacteria. The greatly swollen nucleus destroys its host cell and the bacteria are released after the nuclear membrane bursts. Intriguingly, the only nuclei that were never infected by “ Ca . E. bathymodioli” were those of the gill bacteriocytes. These cells contain the symbiotic sulfur‐ and methane‐oxidizing bacteria, suggesting that the mussel symbionts can protect their host nuclei against the parasite. Phylogenetic analyses showed that the “ Ca . E. bathymodioli” belongs to a monophyletic clade of Gammaproteobacteria associated with marine metazoans as diverse as sponges, corals, bivalves, gastropods, echinoderms, ascidians and fish. We hypothesize that many of the sequences from this clade originated from intranuclear bacteria, and that these are widespread in marine invertebrates.