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Abstract Methylotrophic planktonic bacteria fulfill a particular role in the carbon cycle of lakes via the turnover of single-carbon compounds. We studied two planktonic freshwater lineages (LD28 and PRD01a001B) affiliated with Methylophilaceae (Betaproteobacteria) in Lake Zurich, Switzerland, by a combination of molecular and cultivation-based approaches. Their spatio-temporal distribution was monitored at high resolution (n=992 samples) for 4 consecutive years. LD28 methylotrophs constituted up to 11 × 107 cells l−1 with pronounced peaks in spring and autumn–winter, concomitant with blooms of primary producers. They were rare in the warm water layers during summer but abundant in the cold hypolimnion, hinting at psychrophilic growth. Members of the PRD01a001B lineage were generally less abundant but also had maxima in spring. More than 120 axenic strains from these so far uncultivated lineages were isolated from the pelagic zone by dilution to extinction. Phylogenetic analysis separated isolates into two distinct genotypes. Isolates grew slowly (μmax=0.4 d−1), were of conspicuously small size, and were indeed psychrophilic, with higher growth yield at low temperatures. Growth was enhanced upon addition of methanol and methylamine to sterile lake water. Genomic analyses of two strains confirmed a methylotrophic lifestyle with a reduced set of genes involved in C1 metabolism. The very small and streamlined genomes (1.36 and 1.75 Mb) shared several pathways with the marine OM43 lineage. As the closest described taxa (Methylotenera sp.) are only distantly related to either set of isolates, we propose a new genus with two species, that is, ‘Candidatus Methylopumilus planktonicus’ (LD28) and ‘Candidatus Methylopumilus turicensis’ (PRD01a001B).

A novel anoxygenic photoheterotrophic member of the phylum Acidobacteria , Chloracidobacterium thermophilum strain B sp. nov., was isolated from a cyanobacterial enrichment culture derived from microbial mats associated with Octopus Spring, Yellowstone National Park, WY. C. thermophilum sp. nov. was a Gram-stain-negative rod (diameter, approximately 0.8–1.0 µm; variable length, approximately 2.5 µm), which formed greenish-brown liquid suspension cultures. It was a moderately thermophilic microaerophile and grew in a defined medium at 51 °C (Topt; range 44 to 58 °C) and in the pH range 5.5 to 9.5 (pHopt = ~7.0). The DNA G+C content was 61.3 mol%, and phylogenetic analysis, based on the 16S rRNA sequence, showed that C. thermophilum sp. nov. belongs to subdivision 4 ( Acidobacteriaceae ) of the Acidobacteria . C. thermophilum sp. nov. was unable to synthesize branched-chain amino acids, l-lysine, and vitamin B12, which were required for growth. Although the organism lacked genes/enzymes for autotrophic carbon fixation, bicarbonate was required. Growth was stimulated by other amino acids and 2-oxoglutarate. Cells produced chlorosomes containing a diverse mixture of bacteriochlorophyll (BChl) c derivatives, and additionally, synthesized BChl a P, Chl a PD, and Zn-BChl a′P, which occurred in type-1 homodimeric reaction centres. The carotenoids included echinenone, canthaxanthin, lycopene, γ-carotene and β-carotene. C. thermophilum sp. nov. produced iso-diabolic acid as its major fatty acid and synthesized three hopanoids (diploptene, bacteriohopanetetrol and bacteriohopanetetrol cyclitol ether). Based upon its phenotypic and genotypic properties, the name Chloracidobacterium thermophilum gen. nov., sp. nov. is proposed for this isolate; the type strain is C. thermophilum strain BT (ATCC BAA-2647 = JCM 30199).

Phytoplasmas are a diverse but phylogenetically coherent group of cell-wall-less bacteria affiliated with the class Mollicutes . Due to difficulties in establishing axenic culture, phytoplasmas were assigned to a provisional genus, ‘Candidatus Phytoplasma’, and the genus was embraced within the order Acholeplasmatales . However, phytoplasmas differ significantly from species of the genus Acholeplasma in their habitat specificities, modes of life, metabolic capabilities, genomic architectures, and phylogenetic positions. This communication describes the unique ecological, nutritional, biochemical, genomic and phylogenetic properties that distinguish phytoplasmas from species of the genus Acholeplasma and all other taxa in the class Mollicutes . Since such distinguishing properties of the phytoplasmas are not referable to the descriptions of the order Acholeplasmatales and of all other existing orders, namely Mycoplasmatales , Entomoplasmatales and Anaeroplasmatales , this communication raises the question of whether ‘ Candidatus Phytoplasma ’ should be retained in the order Acholeplasmatales or whether a novel provisional order and family should be created to accommodate the genus ‘ Ca. Phytoplasma ’.

A new Gram-staining-positive, non-sporulating, mesophilic, amino acid-degrading anaerobic bacterium, designated strain OTA 102T, was isolated from an anaerobic sequencing batch reactor treating wastewater from cooking tuna. The cells were curved rods (0.6–2.5×0.5 µm) and occurred singly or in pairs. The strain was motile by means of one lateral flagellum. Strain OTA 102T grew at temperatures between 30 and 45 °C (optimum 40 °C), between pH 6.0 and 8.4 (optimum pH 7.2) and NaCl concentrations between 1 and 5 % (optimum 2 %, w/v). Strain OTA 102T required yeast extract for growth. Serine, threonine, glycine, cysteine, citrate, fumarate, α-ketoglutarate and pyruvate were fermented. When co-cultured with Methanobacterium formicicum as the hydrogen scavenger, strain OTA 102T oxidized alanine, valine, leucine, isoleucine, aspartate, tyrosine, methionine, histidine and asparagine. The genomic DNA G+C content of strain OTA 102T was 41.7 mol%. The main fatty acid was iso-C15 : 0. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain OTA 102T was related to Aminobacterium colombiense and Aminobacterium mobile (95.5 and 95.2 % similarity, respectively), of the phylum Synergistetes . On the basis of phylogenetic, genetic and physiological characteristics, strain OTA 102T is proposed to represent a novel species of the genus Aminobacterium , Aminobacterium thunnarium sp. nov. The type strain is OTA 102T ( = DSM 27500T = JCM 19320T).

A phylogenetically novel proteobacterium, strain Shr3T, was isolated from sand gravels collected from the eastern margin of the Sahara Desert. The isolation strategy targeted bacteria filterable through 0.2-µm-pore-size filters. Strain Shr3T was determined to be a Gram-negative, aerobic, non-motile, filamentous bacterium. Oxidase and catalase reactions were positive. Strain Shr3T showed growth on R2A medium, but poor or no growth on nutrient agar, trypticase soy agar and standard method agar. The major isoprenoid quinone was menaquinone-7. The dominant cellular fatty acids detected were C16 : 1ω5c and C16 : 0, and the primary hydroxy acid present was C12 : 0 3-OH. The DNA G+C content was 54.0 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain Shr3T was affiliated with an uncultivated lineage of the phylum Proteobacteria ; the nearest known type strain, with 83 % sequence similarity, was Desulfomicrobium orale DSM 12838T in the class Deltaproteobacteria . The isolate and closely related environmental clones formed a novel class-level clade in the phylum Proteobacteria with high bootstrap support (96–99 %). Based on these results, the novel class Oligoflexia classis nov. in the phylum Proteobacteria and the novel genus and species Oligoflexus tunisiensis gen. nov., sp. nov. are proposed for strain Shr3T, the first cultivated representative of the Oligoflexia. The type strain of Oligoflexus tunisiensis is Shr3T ( = JCM 16864T = NCIMB 14846T). We also propose the subordinate taxa Oligoflexales ord. nov. and Oligoflexaceae fam. nov. in the class Oligoflexia.

A thermotolerant, Gram-strain-negative, non-spore-forming and strictly aerobic bacterium, designated GU51T, was isolated from Guhai hot spring in Jimsar county, Xinjiang province, north-west China. Each cell of strain GU51T consisted of an oval body and two symmetrical long (3–6 µm) prosthecae. The strain moved by polar flagellum. Oxidase and catalase were produced. Strain GU51T grew within the ranges of 37–65 °C (optimum 48–50 °C), 0.5–7.5 % (w/v) NaCl (optimum 2–3 %) and pH 6.0–9.0 (optimum pH 7.5). The major respiratory quinone detected was ubiquinone 10 (U-10) and the genomic DNA G+C content was 66.7±0.4 mol%. Major fatty acids (>5 %) were C16 : 0, C18 : 1ω7c and 11-methyl C18 : 1ω7c. The polar lipids consisted of diphosphatidylglycerol, five glycolipids, phosphatidylglycerol and an unknown phospholipid. Phylogenetic analysis showed the closest relatives of strain GU51T were members of the genus Parvularcula with 92.3 % 16S rRNA gene sequence similarity. On the basis of this polyphasic taxonomic characterization, it is suggested that strain GU51T represents a novel species of a new genus in the family ‘ Parvularculaceae ’, for which the name Amphiplicatus metriothermophilus gen. nov., sp. nov. is proposed. The type strain of the type species is GU51T ( = CGMCC 1.12710T = JCM 19779T).

A thermophilic, filamentous, heterotrophic bacterium, designated strain JAD2T, a member of an as-yet uncultivated lineage that is present and sometimes abundant in some hot springs worldwide, was isolated from sediment of Great Boiling Spring in Nevada, USA. Cells had a mean diameter of 0.3 µm and length of 4.0 µm, and formed filaments that typically ranged in length from 20 to 200 µm. Filaments were negative for the Gram stain reaction, spores were not formed and motility was not observed. The optimum temperature for growth was 72.5–75 °C, with a range of 67.5–75 °C, and the optimum pH for growth was 6.75, with a range of pH 6.5–7.75. Peptone, tryptone or yeast extract were able to support growth when supplemented with vitamins, but no growth was observed using a variety of defined organic substrates. Strain JAD2T was microaerophilic and facultatively anaerobic, with optimal growth at 1 % (v/v) O2 and an upper limit of 8 % O2. The major cellular fatty acids (>5 %) were C16 : 0, C19 : 0, C18 : 0, C20 : 0 and C19 : 1. The genomic DNA G+C content was 69.3 mol%. Phylogenetic and phylogenomic analyses using sequences of the 16S rRNA gene and other conserved genes placed JAD2T within the phylum Chloroflexi , but not within any existing class in this phylum. These results indicate that strain JAD2T is the first cultivated representative of a novel lineage within the phylum Chloroflexi , for which we propose the name Thermoflexus hugenholtzii gen. nov., sp. nov., within Thermoflexia classis nov., Thermoflexales ord. nov. and Thermoflexaceae fam. nov. The type strain of Thermoflexus hugenholtzii is JAD2T ( = JCM 19131T = CCTCC AB-2014030T).

Anaplasma platys ( Apl), ‘ Candidatus Mycoplasma haemominutum’ ( CMh), Bartonella henselae ( Bh) and Bartonella koehlerae ( Bk) were confirmed by polymerase chain reaction (PCR) amplification and DNA sequencing in a cat diagnosed with multiple myeloma. Other inconsistently documented hematologic abnormalities included anemia, thrombocytopenia, eosinophilia and hypoglycemia. Persistent Apl infection was confirmed for the first time in a North American cat by sequencing three bacterial genes ( 16S rRNA, p44 and GroEL) in peripheral blood samples collected 100 days apart. Following doxycycline treatment for Apl, multiple myeloma was diagnosed based upon a monoclonal gammopathy and splenic plasmacytosis, and the cat was treated with melphalan, chlorambucil and prednisolone. Apl DNA was not amplified from post-treatment blood samples and the hyperglobulinemia resolved temporarily following chemotherapy. Retrospective PCR analysis of stored DNA extracts identified CMh, Bk and Bh infections. Retrospective PCR for antigen receptor rearrangements (PARR) of splenic aspirates did not confirm B- or T-cell clonality. Co-infection with multiple vector-borne pathogens should be a diagnostic consideration in cats with chronic hypergammaglobulinemia, monoclonal gammopathy and splenic plasmacytosis.