Ohashi, Akiyoshi

Abstract Candidatus Patescibacteria, also known as candidate phyla radiation (CPR), including the class‐level uncultured clade JAEDAM01 (formerly a subclass of Gracilibacteria/GN02/BD1‐5), are ubiquitous in activated sludge. However, their characteristics and relationships with other organisms are largely unknown. They are believed to be episymbiotic, endosymbiotic or predatory. Despite our understanding of their limited metabolic capacity, their precise roles remain elusive due to the difficulty in cultivating and identifying them. In previous research, we successfully recovered high‐quality metagenome‐assembled genomes (MAGs), including a member of JAEDAM01 from activated sludge flocs. In this study, we designed new probes to visualize the targeted JAEDAM01‐associated MAG HHAS10 and identified its host using fluorescence in situ hybridization (FISH). The FISH observations revealed that JAEDAM01 HHAS10‐like cells were located within dense clusters of Zoogloea , and the fluorescence brightness of zoogloeal cells decreased in the vicinity of the CPR cells. The Zoogloea MAGs possessed genes related to extracellular polymeric substance biosynthesis, floc formation and nutrient removal, including a polyhydroxyalkanoate (PHA) accumulation pathway. The JAEDAM01 MAG HHAS10 possessed genes associated with type IV pili, competence protein EC and PHA degradation, suggesting a Zoogloea ‐dependent lifestyle in activated sludge flocs. These findings indicate a new symbiotic relationship between JAEDAM01 and Zoogloea .

Candidatus Saccharibacteria is a well-described candidate phylum that has not been successfully isolated. Nevertheless, its presence was suggested by 16S rRNA gene sequencing data, and it is frequently detected in natural environments and activated sludge. Because pure culture representatives of Candidatus Saccharibacteria are lacking, the specificity of primers for the determination of their abundance and diversity should be carefully evaluated. In this study, eight Candidatus Saccharibacteria-specific primers were selected from previous studies and evaluated for their coverage against a public database, annealing temperature of the combined primer sets, as well as their utilization to determine the detection frequencies and phylogenetic diversity by cloning analysis, and in quantification by quantitative polymerase chain reaction (PCR). Among the eight primers, four primers (TM7314F, TM7580F, TM7-910R, and TM7-1177R) showed high coverage. Cloning analysis showed that four primer sets (TM7314F and TM7-910R, TM7314F and TM7-1177R, TM7580F and TM7-910R, and TM7580F and TM7-1177R) yielded high detection frequencies for Candidatus Saccharibacteria in activated sludge from a wastewater treatment plant in Higashihiroshima City, Japan. Quantitative PCR results indicated that the primer set containing TM7314F and TM7-910R was superior for the specific detection of Candidatus Saccharibacteria in activated sludge.

ABSTRACT The phylogenetic affiliation and physiological characteristics (e.g., K s and maximum specific growth rate [μ max ]) of an anaerobic ammonium oxidation (anammox) bacterium, “ Candidatus Scalindua sp.,” enriched from the marine sediment of Hiroshima Bay, Japan, were investigated. “ Candidatus Scalindua sp.” exhibits higher affinity for nitrite and a lower growth rate and yield than the known anammox species.

One thermophilic (strain IMO-1T) and two mesophilic (strains KIBI-1Tand YMTK-2T) non-spore-forming, non-motile, Gram-negative, multicellular filamentous micro-organisms, which were previously isolated as members of the tentatively named class ‘Anaerolineae’ of the phylumChloroflexi, were characterized. All isolates were strictly anaerobic micro-organisms. The length of the three filamentous isolates was greater than 100 μm and the width was 0.3–0.4 μm for strain IMO-1T, 0.4–0.5 μm for strain KIBI-1Tand thinner than 0.2 μm for strain YMTK-2T. Strain IMO-1Tcould grow at pH 6.0–7.5 (optimum growth at pH 7.0). The optimal temperature for growth of strain IMO-1Twas around 50 °C (growth occurred between 42 and 55 °C). Growth of the mesophilic strains KIBI-1Tand YMTK-2Toccurred at pH 6.0–7.2 with optimal growth at pH 7.0. Both of the mesophilic strains were able to grow in a temperature range of 25–50 °C with optimal growth at around 37 °C. Yeast extract was required for growth of all three strains. All the strains could grow with a number of carbohydrates in the presence of yeast extract. The G+C contents of the DNA of strains IMO-1T, KIBI-1Tand YMTK-2Twere respectively 53.3, 59.5 and 48.2 mol%. Major fatty acids for thermophilic strain IMO-1Twere anteiso-C17 : 0, iso-C15 : 0, C16 : 0and anteiso-C15 : 0, whereas those for mesophilic strains KIBI-1Tand YMTK-2Twere branched C14 : 0, iso-C15 : 0, C16 : 0and branched C17 : 0, and branched C17 : 0, C16 : 0, C14 : 0and C17 : 0, respectively. Detailed phylogenetic analyses based on their 16S rRNA gene sequences indicated that the isolates belong to the class-level taxon ‘Anaerolineae’ of the bacterial phylumChloroflexi, which for a long time had been considered as a typical uncultured clone cluster. Their morphological, physiological, chemotaxonomic and genetic traits strongly support the conclusion that these strains should be described as three novel independent taxa in the phylumChloroflexi. Here,Anaerolinea thermolimosasp. nov. (type strain IMO-1T=JCM 12577T=DSM 16554T),Levilinea saccharolyticagen. nov., sp. nov. (type strain KIBI-1T=JCM 12578T=DSM 16555T) andLeptolinea tardivitalisgen. nov., sp. nov. (type strain YMTK-2T=JCM 12579T=DSM 16556T) are proposed. In addition, we formally propose to subdivide the tentative class-level taxon ‘Anaerolineae’ intoAnaerolineaeclassis nov. andCaldilineaeclassis nov. We also propose the subordinate taxaAnaerolinealesord. nov.,Caldilinealesord. nov.,Anaerolineaceaefam. nov. andCaldilineaceaefam. nov.