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ABSTRACT To characterize intracellular gram-negative bacteria associated with epitheliocystis in farmed Atlantic salmon ( Salmo salar ), gills with proliferative lesions were collected for histopathology, conventional transmission and immunoelectron microscopy, in situ hybridization, and DNA extraction during epitheliocystis outbreaks in Ireland and Norway in 1999 and 2000, respectively, and compared by ultrastructure and immunoreactivity to nonproliferative gills from Ireland archived in 1995. Genomic DNA from proliferative gills was used to amplify 16S ribosomal DNA (rDNA) for molecular phylogenetic analyses. Epitheliocystis inclusions from proliferative gills possessed variably elongate reticulate bodies, examples of binary fission, and vacuolated and nonvacuolated intermediate bodies, whereas inclusions in nonproliferative gills had typical chlamydial developmental stages plus distinctive head-and-tail cells. Immunogold processing using anti-chlamydial lipopolysaccharide antibody labeled reticulate bodies from proliferative and nonproliferative gills. 16S rDNA amplified directly from Irish (1999) and Norwegian (2000) gill samples demonstrated 99% nucleotide identity, and riboprobes transcribed from cloned near-full-length 16S rDNA amplicons from Norwegian gills hybridized with inclusions in proliferative lesions from Irish (1999) and Norwegian (2000) sections. A 1,487-bp consensus 16S rRNA gene sequence representing the chlamydia-like bacterium (CLB) from proliferative gills had the highest percent nucleotide identity with endosymbionts of Acanthamoeba spp. (order Chlamydiales ). Molecular phylogenetic relationships inferred from 16S rRNA gene sequences using distance and parsimony indicated that the CLB from proliferative gills branched with members of the order Chlamydiales . “ Candidatus Piscichlamydia salmonis” is proposed for the CLB associated with epitheliocystis from proliferative gills of Atlantic salmon, which exhibits developmental stages different from those identified in nonproliferative gills.

ABSTRACT “ Candidatus Glomeribacter gigasporarum” is an endocellular β-proteobacterium present in the arbuscular mycorrhizal (AM) fungus Gigaspora margarita . We established a protocol to isolate “ Ca . Glomeribacter gigasporarum” from its host which allowed us to carry out morphological, physiological, and genomic investigations on purified bacteria. They are rod shaped, with a cell wall typical of gram-negative bacteria and a cytoplasm rich in ribosomes, and they present no flagella or pili. Isolated bacteria could not be grown in any of the 19 culture media tested, but they could be kept alive for up to 4 weeks. PCR-based investigations of purified DNA from isolated bacteria did not confirm the presence of all genes previously assigned to “ Ca . Glomeribacter gigasporarum.” In particular, the presence of nif genes could not be detected. Pulsed-field gel electrophoresis analyses allowed us to estimate the genome size of “ Ca . Glomeribacter gigasporarum” to approximately 1.4 Mb with a ca. 750-kb chromosome and a 600- to 650-kb plasmid. This is the smallest genome known for a β-proteobacterium. Such small genome sizes are typically found in endocellular bacteria living permanently in their host. Altogether, our data suggest that “ Ca . Glomeribacter gigasporarum” is an ancient obligate endocellular bacterium of the AM fungus G. margarita .

A novel bacterium that infects laboratory rats was isolated from wild Rattus norvegicus rats in Japan. Transmission electron microscopy of the spleen tissue revealed small cocci surrounded by an inner membrane and a thin, rippled outer membrane in a membrane-bound inclusion within the cytoplasm of endothelial cells. Phylogenetic analysis of the 16S rRNA gene sequence of the bacterium found in R. norvegicus rats and Ixodes ovatus ticks in Japan revealed that the organism represents a novel clade in the family Anaplasmataceae, which includes the Schotti variant found in Ixodes ricinus ticks in the Netherlands and the Ehrlichia-like Rattus strain found in R. norvegicus rats from China. The novel clade was confirmed by phylogenetic analysis of groESL sequences found in R. norvegicus rats and Ixodes ovatus ticks in Japan. No serological cross-reactivity was detected between this bacterium and members of the genera Anaplasma, Ehrlichia or Neorickettsia in the family Anaplasmataceae. It is proposed that this new cluster of bacteria should be designated ‘Candidatus Neoehrlichia mikurensis’.

Intracellular bacteria were observed in the hepatopancreas of the terrestrial isopod Porcellio scaber. Comparative 16S rRNA gene sequence analysis and electron microscopic observations were used to determine the taxonomic position of these intracellular bacteria. Phylogenetic analysis and a complex developmental cycle affiliate these bacteria to the order Chlamydiales, within which they form a distinctive lineage, close to the family Simkaniaceae. They share <92 % 16S rRNA gene sequence similarity with their closest relative and <88 % similarity with other members of the order Chlamydiales. A specific signature oligonucleotide sequence was identified and used as a probe, enabling the identification of intracellular bacteria in infected hepatopancreatic tissue. According to the distinctive morphology of their elementary bodies, which are rod-shaped rather than spherical and contain translucent oblong structures, their genomic properties and their crustacean host, the name ‘Candidatus Rhabdochlamydia porcellionis' is proposed for intracellular bacteria in the hepatopancreas of P. scaber.

A thermophilic, marine, anaerobic, chemolithoautotrophic, sulfate-reducing bacterium, strain CIR29812T, was isolated from a deep-sea hydrothermal vent site at the Kairei vent field on the Central Indian Ridge. Cells were Gram-negative motile rods that did not form spores. The temperature range for growth was 55–80 °C, with an optimum at 70 °C. The NaCl concentration range for growth was 10–35 g l−1, with an optimum at 25 g l−1. The pH range for growth was 6–6·7, with an optimum at approximately pH 6·25. H2and CO2were the only electron donor and carbon source found to support growth of the strain. However, several organic compounds were stimulatory for growth. Sulfate was used as electron acceptor, whereas elemental sulfur, thiosulfate, sulfite, cystine, nitrate and fumarate were not. No fermentative growth was observed with malate, pyruvate or lactate. The phenotypic characteristics of strain CIR29812Twere similar to those ofThermodesulfobacterium hydrogeniphilum, a recently described thermophilic, chemolithoautotrophic sulfate-reducer. However, phylogenetic analyses of the 16S rRNA gene sequences showed that the new isolate was distantly related to members of the familyThermodesulfobacteriaceae(similarity values of less than 90 %). The chemotaxonomic data (fatty acids and polar lipids composition) also indicated that strain CIR29812Tcould be distinguished fromThermodesulfobacterium commune, the type species of the type genus of the familyThermodesulfobacteriaceae. Finally, the G+C content of the genomic DNA of strain CIR29812T(46·0 mol%) was not in the range of values obtained for members of this family. On the basis of phenotypic, chemotaxonomic and genomic features, it is proposed that strain CIR29812Trepresents a novel species of a new genus,Thermodesulfatator, of whichThermodesulfatator indicusis the type species. The type strain is CIR29812T(=DSM 15286T=JCM 11887T).