Publications

4517

ABSTRACTUncultivated bacteria that densely colonize the midgut glands (hepatopancreas) of the terrestrial isopodPorcellio scaber(Crustacea: Isopoda) were identified by cloning and sequencing of their 16S rRNA genes. Phylogenetic analysis revealed that these symbionts represent a novel lineage of theMollicutesand are only distantly related (<82% sequence identity) to members of theMycoplasmatalesandEntomoplasmatales. Fluorescence in situ hybridization with a specific oligonucleotide probe confirmed that the amplified 16S rRNA gene sequences indeed originated from a homogeneous population of symbionts intimately associated with the epithelial surface of the hepatopancreas. The same probe also detected morphotypically identical symbionts in other crinochete isopods. Scanning and transmission electron microscopy revealed uniform spherical bacterial cells without a cell wall, sometimes interacting with the microvilli of the brush border by means of stalk-like cytoplasmic appendages, which also appeared to be involved in cell division through budding. Based on the isolated phylogenetic position and unique cytological properties, the provisional name “CandidatusHepatoplasma crinochetorum” is proposed for this new taxon ofMollicutescolonizing the hepatopancreas ofP. scaber.

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’.

ABSTRACT The bryozoans Bugula neritina and Bugula simplex harbor bacteria in the pallial sinuses of their larvae as seen by electron microscopy. In B. neritina , the bacterial symbiont has been characterized as a gamma-proteobacterium, “ Candidatus Endobugula sertula.” “ Candidatus E. sertula” has been implicated as the source of the bryostatins, polyketides that provide chemical defense to the host and are also being tested for use in human cancer treatments. In this study, the bacterial symbiont in B. simplex larvae was identified by 16S rRNA-targeted PCR and sequencing as a gamma-proteobacterium closely related to and forming a monophyletic group with “ Candidatus E. sertula.” In a fluorescence in situ hybridization, a 16S ribosomal DNA probe specific to the B. simplex symbiont hybridized to long rod-shaped bacteria in the pallial sinus of a B. simplex larva. The taxonomic status “ Candidatus Endobugula glebosa” is proposed for the B. simplex larval symbiont. Degenerate polyketide synthase (PKS) primers amplified a gene fragment from B. simplex that closely matched a PKS gene fragment from the bryostatin PKS cluster. PCR surveys show that the symbiont and this PKS gene fragment are consistently and uniquely associated with B. simplex . Bryostatin activity assays and chemical analyses of B. simplex extracts reveal the presence of compounds similar to bryostatins. Taken together, these findings demonstrate a symbiosis in B. simplex that is similar and evolutionarily related to that in B. neritina .

The symbioses between cellulose-degrading flagellates and bacteria are one of the most fascinating phenomena in the complex micro-ecosystem found in the hindgut of lower termites. However, little is known about the identity of the symbionts. One example is the epibiotic bacteria colonizing the surface of hypermastigote protists of the genusStaurojoenina. By using scanning electron microscopy, it was shown that the whole surface ofStaurojoeninasp. from the termiteNeotermes cubanusis densely covered with long rod-shaped bacteria of uniform size and morphology. PCR amplification of 16S rRNA genes from isolated protozoa and subsequent cloning yielded a uniform collection of clones with virtually identical sequences. Phylogenetic analysis placed them as a new lineage among theBacteroidales, only distantly related to other uncultivated bacteria in the hindgut of other termites, including an epibiont of the flagellateMixotricha paradoxa. The closest cultivated relative wasTannerella forsythensis(<85 % sequence identity). Fluorescencein situhybridization with a newly designed clone-specific oligonucleotide probe confirmed that these sequences belong to the rod-shaped epibionts ofStaurojoeninasp. Transmission electron microscopy confirmed the presence of a Gram-negative cell wall and revealed special attachment sites for the symbionts on the cell envelope of the flagellate host. Based on the isolated phylogenetic position and the specific association with the surface ofStaurojoeninasp., we propose to classify this new taxon ofBacteroidalesunder the provisional name ‘CandidatusVestibaculum illigatum’.

Spartium witches'-broom (SpaWB), buckthorn witches'-broom (BWB) and allocasuarina yellows (AlloY) are witches'-broom and yellows diseases of Spartium junceum (Spanish broom), Rhamnus catharticus (buckthorn) and Allocasuarina muelleriana (Slaty she-oak), respectively. These diseases are associated with distinct phytoplasmas. The SpaWB, BWB and AlloY phytoplasmas share <97·5 % 16S rDNA sequence similarity with each other and with other known phytoplasmas, including the closely related phytoplasmas of the apple proliferation group. Also, the SpaWB, BWB and AlloY phytoplasmas each have a different natural plant host. Based on their unique properties, it is proposed to designate the mentioned phytoplasmas as novel ‘Candidatus’ species under the names ‘Candidatus Phytoplasma spartii’, ‘Candidatus Phytoplasma rhamni’ and ‘Candidatus Phytoplasma allocasuarinae’, respectively.

Five strains with strong chitinolytic activity were isolated from a soil sample collected from southern Taiwan. The strains shared more than 92 % DNA–DNA similarity, indicating membership of the same genospecies. This close relationship was supported by high similarities in fatty acid composition and biochemical characteristics. A 16S rRNA gene sequence analysis indicated that the isolates were members of the class ‘Betaproteobacteria’, in which they formed an individual subline of descent that was distantly related (<94 % similarity) to lineages defined by Formivibrio citricus DSM 6150T and Iodobacter fluviatilis DSM 3764T. On the basis of the phylogenetic and phenotypic distinctness of these novel chitin-degrading organisms, a new genus, Chitinibacter, is proposed, with Chitinibacter tainanensis (type strain, S1T=BCRC 17254T=DSM 15459T) as the type species.

Bermuda grass white leaf (BGWL) is a destructive, phytoplasmal disease of Bermuda grass (Cynodon dactylon). The causal pathogen, the BGWL agent, differs from other phytoplasmas that cluster in the same major branch of the phytoplasma phylogenetic clade in <2·5 % of 16S rDNA nucleotide positions, the threshold for assigning species rank to phytoplasmas under the provisional status ‘Candidatus’. Thus, the objective of this work was to examine homogeneity of BGWL isolates and to determine whether there are, in addition to 16S rDNA, other markers that support delineation of the BGWL agent at the putative species level. Phylogenetic analyses revealed that the 16S rDNA sequences of BGWL strains were identical or nearly identical. Clear differences that support separation of the BGWL agent from related phytoplasmas were observed within the 16S–23S rDNA spacer sequence, by serological comparisons, in vector transmission and in host-range specificity. From these results, it can be concluded that the BGWL phytoplasma is a discrete taxon at the putative species level, for which the name ‘Candidatus Phytoplasma cynodontis' is proposed. Strain BGWL-C1 was selected as the reference strain. Phytoplasmas that are associated with brachiaria white leaf, carpet grass white leaf and diseases of date palms showed 16S rDNA and/or 16S–23S rDNA spacer sequences that were identical or nearly identical to those of the BGWL phytoplasmas. However, the data available do not seem to be sufficient for a proper taxonomic assignment of these phytoplasmas.