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A bacterial strain, designated Sp-1 T , was isolated from the heterotrich ciliate Spirostomum yagiui collected from a reservoir located in Ulsan, Republic of Korea. Cells of Sp-1 T were Gram stain-negative, rod-shaped, non-spore-forming, non-motile and contained poly-β-hydroxybutyrate granules. Phylogenetic analyses based on 16S rRNA gene sequences indicated that Sp-1 T constituted a distinct phylogenetic lineage within different families in the order Rhizobiales with a pairwise sequence similarity of 95 % to the species of the genus Ochrobactrum: Ochrobactrum anthropi ATCC 49188 T and Ochrobactrum cytisi ESC1 T (family Brucellaceae ). The major cellular fatty acids were C 19 : 0 cyclo ω8 c (44.4 %) and C 16 : 0 (32.1 %). The identified sole isoprenoid quinone was ubiquinone-10 (Q-10). The major polar lipids produced were phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, an unidentified aminolipid, two unidentified phospholipids and three unidentified lipids. The genome size was about 5.4 Mbp and the DNA G+C content was 68.2 mol%. Sp-1 T exhibited the highest average nucleotide identity value of 76.6 % and in silico DNA-DNA hybridization value of 22.1 % with Pseudoxanthobacter soli DSM 19599 T (family Xanthobacteraeae ). This strain is distinguishable from closely related members of the order Rhizobiales by its differential phenotypic, chemotaxonomic, genomic and phylogenetic characteristics. On the basis of evidence from polyphasic taxonomic analysis, we concluded that Sp-1 T represents a novel species in a novel genus within the order Rhizobiales , for which the name Segnochrobactrum spirostomi gen. nov., sp. nov. is proposed. The type strain is Sp-1 T (=KCTC 62036 T =JCM 32162 T ). We also describe a novel family, Segnochrobactraceae fam. nov., to encompass the proposed novel genus and species.

We report a discovery of a novel family of anaerobic ciliates, Tropidoatractidae fam. nov. Phylogenetic analyses based on the 18S rRNA gene show that the family Tropidoatractidae corresponds to the previously reported clade of environmental sequences closely related to the lineage consisting of orders Metopida and Clevelandellida. The family comprises two genera, Tropidoatractus and Palmarella, and five species, two of which are newly described herein. Tropidoatractidae are cosmopolitan Metopida with sparse somatic and oral ciliature, deep, cup-like buccal cavity, and hyaline cortex with interkinetal ridges. Moreover, all species occur in two morphotypes, slender and stout. They inhabit microoxic or anoxic freshwater, brackish, and marine sediments and possess anaerobic mitochondrion-related organelles and various prokaryotic symbionts. The discovery of Tropidoatractidae provides valuable information about the evolution of Armophorea and gives us insights to the diversity and ecological preferences of anaerobic ciliates in general., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

The anaerobic free-living ciliate, Trimyema compressum, is known to harbor both methanogenic archaeal and bacterial symbionts in the cytoplasm. To clarify their phylogenetic belongings, a full-cycle rRNA approach was applied to this symbiosis. Phylogenetic analysis showed that the methanogenic symbiont was related to Methanobrevibacter arboriphilicus, which was distantly related to symbionts found in other Trimyema species. This result suggested that Trimyema species do not require very specific methanogenic symbionts, and symbiont replacement could have occurred in the history of Trimyema species. On the other hand, the bacterial symbiont was located near the lineage of the family Syntrophomonadaceae in the phylum Firmicutes. The sequence similarity between the bacterial symbiont and the nearest species was 85%, indicating that bacterial symbionts may be specific to the Trimyema species. The elimination of bacterial symbionts from the ciliate cell by antibiotic treatment resulted in considerably decreased host growth. However, it was not restored by stigmasterol addition (<2 microg ml(-1)), which was different from the previous report that showed that the symbiont-free strain required exogenous sterols for growth. In addition, the decline of host growth was not accompanied by host metabolism shift toward the formation of more reduced products, which suggested that the contribution of bacterial symbionts to the host ciliate was not a dispose of excessive reducing equivalent arising from the host's fermentative metabolism as methanogenic symbionts do. This study showed that bacterial symbionts make a significant contribution to the host ciliate by an unknown function and suggested that interactions between bacterial symbionts and T. compressum are more complicated than hitherto proposed.

The identities and taxonomic diversity of the endosymbiotic methanogens from the anaerobic protozoa Metopus contortus, Metopus striatus, Metopus palaeformis, Trimyema sp. and Pelomyxa palustris were determined by comparative analysis of their 16S ribosomal RNA sequences. Fluorescent oligonucleotide probes were designed to bind to the symbiont rRNA sequences and to provide direct visual evidence of their origins from methanogenic archaea contained within the host cells. Confocal microscopy was used to analyze the morphology of the endosymbionts in whole cells of Metopus palaeformis, Metopus contortus, Trimyema sp, and Cyclidium porcatum. The endosymbionts are taxonomically diverse and are drawn from three different genera; Methanobacterium, Methanocorpusculum and Methanoplanus. In every case the symbionts are closely related to, but different from, free-living methanogens for which sequences are available. It is thus apparent that symbioses have been formed repeatedly and independently. Ciliates which are unrelated to each other (Trimyema sp. and Metopus contortus) may contain symbionts which are closely related, and congeneric ciliates (Metopus palaeformis and M. contortus) may contain symbionts which are distantly related to each other. This suggests that some of the symbiotic associations must be relatively recent. For example, at least one of the symbioses in Metopus must postdate the speciation of M. palaeformis and M. contortus. Despite this, Metopus contortus, Trimyema sp., Cyclidium porcatum and their respective endosymbionts show sophisticated morphological interactions which probably facilitate the exchange of materials between the partners.

The polymerase chain reaction (PCR) was used to amplify small-subunit ribosomal DNA from the anaerobic ciliated protozoon Metopus palaeformis, and from its uncultured endosymbiotic bacteria. This was accomplished directly from total DNA extracted from protozoa without prior isolation or enrichment for symbiont cells. The double-stranded amplification products were precipitated and directly sequenced using the linear PCR reaction. Fluorescent oligonucleotide probes were designed and used in whole-cell hybridizations to provide direct visual evidence that the sequences originated from the host ciliate and from the endosymbiont. Phylogenetic analysis of the Metopus palaeformis sequence consistently placed it as a deep-branching lineage near the root of the ciliate tree. However, the present data were insufficient to resolve the detailed relationship between Blepharisma and Metopus and thus to determine if the heterotrichs are mono- or paraphyletic. Phylogenetic analysis of the symbiont partial sequence clearly demonstrated that it is an archaeobacterium and that it is closely related to, but distinct from, Methanobacterium formicicum.