Novel aerobic anoxygenic phototrophic bacterium Jannaschia pagri sp. nov., isolated from seawater around a fish farm.

The genus Jannaschia is one of the representatives of aerobic anoxygenic phototrophic (AAP) bacteria, which is a strictly aerobic bacterium, producing a photosynthetic pigment bacteriochlorophyll (BChl) a. However, a part of the genus Jannaschia members have not been confirmed the photosynthetic ability. The partly presence of the ability in the genus Jannaschia could suggest the complexity of evolutionary history for anoxygenic photosynthesis in the genus, which is expected as gene loss and/or horizontal gene transfer. Here a novel AAP bacterium designated as strain AI_62T (= DSM 115720 T = NBRC 115938 T), was isolated from coastal seawater around a fish farm in the Uwa Sea, Japan. Its closest relatives were identified as Jannaschia seohaensis SMK-146 T (95.6% identity) and J. formosa 12N15T (94.6% identity), which have been reported to produce BChl a. The genomic characteristic of strain AI_62T clearly showed the possession of the anoxygenic photosynthesis related gene sets. This could be a useful model organism to approach the evolutionary mystery of anoxygenic photosynthesis in the genus Jannaschia. Based on a comprehensive consideration of both phylogenetic and phenotypic characteristics, we propose the classification of a novel species within the genus Jannaschia, designated as Jannaschia pagri sp. nov. The type strain for this newly proposed species is AI_62T (= DSM 115720 T = NBRC 115938 T).

Proposal of a novel subspecies: Alicyclobacillus hesperidum subspecies aegles.

Alicyclobacillus sp. DSM 11985T was isolated from geothermal soil but had not yet been classified at the species level. The strain produced guaiacol, which is of interest from the viewpoint of food spoilage in the food industry. 16S rRNA gene sequence analysis revealed that strain DSM 11985T was closely related (99.6 % similarity) to Alicyclobacillus hesperidum DSM 12489T. However, strains of A. hesperidum did not produce guaiacol; therefore, we performed the taxonomic characterization of strain DSM 11985T. The results showed that strain DSM 11985T and strains of A. hesperidum showed different phenotypic characteristics in biochemical/physiological tests including guaiacol production. Average nucleotide identity values between strain DSM 11985T and strain DSM 12489T were 95.4-95.9 %, and the in silico DNA-DNA hybridization value using the Genome-to-Genome Distance Calculator between strains DSM 11985T and DSM 12489T was 65.5 %. These values showed that strain DSM 11985T was genetically closely related but separated from strains of A. heparidum. From the above results, a novel subspecies of A. hesperidum, named Alicyclobacillus hesperidum subsp. aegles subsp. nov. is proposed. The type strain is DSM 11985T (=FR-12T=NBRC 113041T).

Flavobacterium okayamense sp. nov. isolated from surface seawater.

Strain KK2020170T, a Gram-stain negative, yellow colony-forming bacterium, was isolated from surface seawater sampled in Kojima Bay, Okayama, Japan. Phylogenetic analysis based on the 16S rRNA gene revealed that strain KK2020170T belongs to the genus Flavobacterium, with Flavobacterium haoranii LQY-7T (98.1% similarity) being its closest relative, followed by Flavobacterium sediminis MEBiC07310T (96.9%) and Flavobacterium urocaniciphilum YIT 12746T (96.0%). Whole-genome shotgun sequencing showed that strain KK2020170T, when paralleled with F. haoranii LQY-7 T, had 81.3% average nucleotide identity, and 24.6% in silico DNA-DNA hybridization values, respectively. The DNA G + C content of strain KK2020170T was 31.1 mol%. The most abundant fatty acids (> 10%) of strain KK2020170T were iso-C15: 0, iso-C17: 0 3-OH and iso-C15: 1 G. The dominant respiratory quinone of the strain was menaquinone MK-6. Based on the phylogenetic and phenotypic analysis results, we propose that strain KK2020170T represents a novel species, for which the name Flavobacterium okayamense sp. nov. has been proposed. The type strain is KK2020170T (= ATCC TSD-280 T = NBRC 115344 T).

Roseomonas fluvialis sp. nov., an aerobic bacteriochlorophyll a-containing freshwater bacterium isolated from river epilithic biofilm.

A strictly aerobic bacteriochlorophyll a-containing alphaproteobacterium, designated strain S08T, was isolated from a biofilm sampled at Tama River in Japan. The non-motile and rod-shaped cells formed pink-beige pigmented colonies on agar plates containing organic compounds and showed in vivo absorption maxima at 798 and 866 nm in the near-infrared region, typical for the presence of bacteriochlorophyll a. The new bacterial isolate is Gram-negative, oxidase-negative and catalase-positive. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain S08T was closely related to species in the genus Roseomonas. The closest phylogenetic relative of strain S08T was Roseomonas lacus TH-G33T (98.2 % sequence similarity). The major cellular fatty acids were C16 : 0, C18 : 1 2-OH and summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c). The predominant respiratory quinone was ubiquinone-9. The major polar lipids contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and an aminolipid. The G+C content of the genomic DNA was 70.6 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between strain S08T and the related Roseomonas type strains were all far lower than the cut-off value for the delineation of species. The results of polyphasic comparisons showed that strain S08T was clearly distinguishable from other members of the genus Roseomonas. Therefore, we propose a new species in the genus Roseomonas, namely, Roseomonas fluvialis sp. nov. The type strain is S08T (=DSM 111902T=NBRC 112025T).

Motility Assays of Chloroflexus.

Chloroflexus is a thermophilic, filamentous, gliding bacterium. Its multicellular filaments of several hundred micrometer length move straightforward at a speed of approximately 1-3 µm/s and occasionally reverse the moving direction. In liquid media, filaments glide on each other to form cell aggregates without tight adhesion. The molecular machinery on the cell surface that forces the gliding movement has not yet been identified. Here, we describe the cultivation methods to characterize the gliding motility of Chlroflexus and the microscopic assays to determine its gliding speed, reversal frequency, and cell-surface movements.

Coprobacter secundus subsp. similis subsp. nov. and Solibaculum mannosilyticum gen. nov., sp. nov., isolated from human feces.

An obligately anaerobic, Gram-stain-negative, rod-shaped bacterium, designated strain 2CBH44T , was isolated from the fecal sample of a healthy Japanese man. This strain was initially assigned as a novel species of the genus Coprobacter based on the 16S rRNA gene sequence similarities compared with other Coprobacter species. The 16S rRNA gene sequence analysis revealed strain 2CBH44T had relatively low 16S rRNA gene sequence similarity (97.5%) to Coprobacter secundus 177T . However, strain 2CBH44T showed 96.9% average nucleotide identity value with C. secundus 177T , indicating that strain 2CBH44T and C. secundus 177T belong to the same species. On the other hand, the digital DNA-DNA hybridization value between strain 2CBH44T and C. secundus 177T was 73.5%, indicating that strain 2CBH44T is a subspecies of C. secundus. Another anaerobic, Gram-stain-variable, rod-shaped bacterium, designated strain 12CBH8T , was also isolated from human feces. Strain 12CBH8T had significantly low 16S rRNA gene sequence similarities (<92.0%) to the validated bacterial species within the family Oscillospiraceae. The percentage of conserved protein values between the genome of strain 12CBH8T and that of the validated related taxa were <50%, suggesting that strain 12CBH8T belongs to a novel genus. On the basis of the collected data, strain 2CBH44T represents a novel subspecies of C. secundus, for which the name Coprobacter secundus subsp. similis subsp. nov. (type strain 2CBH44T = JCM 34079T = DSM 111570T ) is proposed. Strain 12CBH8T represents a novel species of a novel genus, for which the name Solibaculum mannosilyticum gen. nov., sp. nov. (type strain 12CBH8T = JCM 34081T = DSM 111571T ) is proposed.

Fluviibacter phosphoraccumulans gen. nov., sp. nov., a polyphosphate-accumulating bacterium of Fluviibacteraceae fam. nov., isolated from surface river water.

Three aerobic, Gram-stain-negative, non-motile, rod-shaped bacteria, designated as strains SHINM1T, ICHIJ1 and ICHIAU1, were isolated from surface river water (Saitama Prefecture, Japan). Phylogenetic analyses based on 16S rRNA and 40 marker gene sequences revealed that the strains formed a distinct phylogenetic lineage within the order Rhodocyclales. The three strains shared 100 % 16S rRNA gene similarity. Growth occurred at 15-30 °C and pH 6.0-9.5, but not in the presence of ≥1.0 % (w/v) NaCl. The isolates stained positive for intracellular polyphosphate granules. The major cellular fatty acids were C16 : 0, summed feature 2 (C12 : 1 aldehyde and/or iso-C16 : 1 I and/or C14 : 0 3-OH), summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c) and summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c). The major polar lipids were phosphatidylethanolamine and an unidentified phospholipid. The predominant quinone system of strain SHINM1T was ubiquinone-8 and its DNA G+C content was 56.7 mol%. Genome sequencing of the three isolates revealed a genome size of 2.29-2.43 Mbp and average nucleotide identity by orthology values of ≥98.9 %. Based on the results of phenotypic and phylogenetic analyses, strains SHINM1T, ICHIJ1 and ICHIAU1 represent a novel species of a new genus, for which the name Fluviibacter phosphoraccumulans gen. nov., sp. nov. is proposed, within a new family, Fluviibacteraceae fam. nov. of the order Rhodocyclales. The type strain is SHINM1T (=JCM 32071T=NCIMB 15105T).

Cell Plasticity and Genomic Structure of a Novel Filterable Rhizobiales Bacterium that Belongs to a Widely Distributed Lineage.

Rhizobiales bacterium strain IZ6 is a novel filterable bacterium that was isolated from a suspension filtrate (<0.22 µm) of soil collected in Shimane Prefecture, western Japan. Additional closely related isolates were recovered from filterable fractions of terrestrial environmental samples collected from other places in Japan; the Gobi Desert, north-central China; and Svalbard, Arctic Norway. These findings indicate a wide distribution of this lineage. This study reports the cell variation and genomic structure of IZ6. When cultured at lower temperatures (4 °C and 15 °C), this strain contained ultra-small cells and cell-like particles in the filtrate. PacBio sequencing revealed that this chromosome (3,114,641 bp) contained 3150 protein-coding, 51 tRNA, and three rRNA genes. IZ6 showed low 16S rRNA gene sequence identity (<97%) and low average nucleotide identity (<76%) with its closest known relative, Flaviflagellibacter deserti. Unlike the methylotrophic bacteria and nitrogen-fixing bacteria in related genera, there were no genes that encoded enzymes for one-carbon-compound utilization and nitrogen fixation in the IZ6 genome; the genes related to nitrate and nitrite reductase are retained and those related to the cell membrane function tend to be slightly enriched in the genome. This genomic information helps elucidate the eco-physiological function of a phenotypically heterogeneous and diverse Rhizobiales group.

Amedibacterium intestinale gen. nov., sp. nov., isolated from human faeces, and reclassification of Eubacterium dolichum Moore et al. 1976 (Approved Lists 1980) as Amedibacillus dolichus gen. nov., comb. nov.

Four strains (9CBEGH2T, 9BBH35, 6BBH38 and 6EGH11) of Gram-stain-positive, obligately anaerobic, rod-shaped bacteria were isolated from faecal samples from healthy Japanese humans. The results of 16S rRNA gene sequence analysis indicated that the four strains represented members of the family Erysipelotrichaceae and formed a monophyletic cluster with 'Absiella argi' strain N6H1-5 (99.4% sequence similarity) and Eubacterium sp. Marseille-P5640 (99.3 %). Eubacterium dolichum JCM 10413T (94.2 %) and Eubacterium tortuosum ATCC 25548T (93.7 %) were located near this monophyletic cluster. The isolates, 9CBEGH2T, 'A. argi' JCM 30884 and Eubacterium sp. Marseille-P5640 shared 98.7-99.1% average nucleotide identity (ANI) with each other. Moreover, the in silico DNA-DNA hybridization (DDH) values among three strains were 88.4-90.6%, indicating that these strains represent the same species. Strain 9CBEGH2T showed 21.5-24.1 % in silico DDH values with other related taxa. In addition, the ANI values between strain 9CBEGH2T and other related taxa ranged from 71.2 % to 73.5 %, indicating that this strain should be considered as representing a novel species on the basis of whole-genome relatedness. Therefore, we formally propose a novel name for 'A. argi' strains identified because the name 'A. argi' has been effectively, but not validly, published since 2017. On the basis of the collected data, strain 9CBEGH2T represents a novel species of a novel genus, for which the name Amedibacterium intestinale gen. nov., sp. nov. is proposed. The type strain of A. intestinale is 9CBEGH2T (=JCM 33778T=DSM 110575T).

Gliding motility driven by individual cell-surface movements in a multicellular filamentous bacterium Chloroflexus aggregans.

Chloroflexus aggregans is an unbranched multicellular filamentous bacterium having the ability of gliding motility. The filament moves straightforward at a constant rate, ∼3 µm sec(-1) on solid surface and occasionally reverses the moving direction. In this study, we successfully detected movements of glass beads on the cell-surface along long axis of the filament indicating that the cell-surface movement was the direct force for gliding. Microscopic analyses found that the cell-surface movements were confined to a cell of the filament, and each cell independently moved and reversed the direction. To understand how the cellular movements determine the moving direction of the filament, we proposed a discrete-time stochastic model; sum of the directions of the cellular movements determines the moving direction of the filament only when the filament pauses, and after moving, the filament keeps the same directional movement until all the cells pause and/or move in the opposite direction. Monte Carlo simulation of this model showed that reversal frequency of longer filaments was relatively fixed to be low, but the frequency of shorter filaments varied widely. This simulation result appropriately explained the experimental observations. This study proposed the relevant mechanism adequately describing the motility of the multicellular filament in C. aggregans.

Secreted protease mediates interspecies interaction and promotes cell aggregation of the photosynthetic bacterium Chloroflexus aggregans.

Interspecies interactions were studied in hot spring microbial mats where diverse species of bacterial cells are densely packed. The anoxygenic photosynthetic bacterium, Chloroflexus aggregans, has been widely found in the microbial mats as a major component in terrestrial hot springs in Japan at the temperature from 50 to 70°C. C. aggregans shows cellular motility to form a microbial mat-like dense cell aggregate. The aggregating ability of C. aggregans was affected by another bacterial species, strain BL55a (related to Bacillus licheniformis) isolated from the microbial mats containing C. aggregans. Cell aggregation rate of C. aggregans was promoted by the addition of culture supernatants of strain BL55a. Similar effects were also detected from other bacterial isolates, specifically Geobacillus sp. and Aeribacillus sp. Protease activity was detected from the culture supernatants from all of these isolates. The promoting effect of strain BL55a was suppressed by a serine protease inhibitor, phenylmethylsulfonyl fluoride. A purified serine protease, subtilisin obtained from B. licheniformis, showed a promoting effect on the cell aggregation. These results suggest that an extracellular protease, secreted from co-existing bacterial species promoted the aggregating motility of C. aggregans. This is the first report that exogenous protease affects bacterial cellular motility.