Olsenella intestinalis sp. nov., isolated from cow feces

A Gram-stain-negative, anaerobic, non-motile, rod-shaped bacterium, designated as BGYT1T, was isolated from the feces of a cow in Andong, Republic of Korea. It was studied using a polyphasic method to determine its taxonomic position. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain BGYT1T formed a lineage within the genus Olsenella and was most closely related to O. umbonate KCTC 15140T (98.2%). The complete genome sequence of strain BGYT1T was 2,476,083 bp long with a G + C content of 66.9 mol% and contained 1835 genes and 8 contigs. The N50 value was 604,117 bp. There were 50 tRNAs, 6 rRNAs (5S, 16S, 23S), 1778 CDSs and 2 BGCs and 1 tmRNA. The values for ANI (76.8%), AAI (67.3%), and dDDH (22.2%) compared to the closest related species were all below the threshold for bacterial species delineation. In addition, genes encoding the cell wall degrading enzymes such as chitinases, β-1,3 glucanases, and proteases were also detected. The strain was able to grow at pH 6.0–8.0 (optimum, pH 7.0), in the presence of 0.5–1.5% NaCl (optimum, 0.5%, w/v) and at the temperature range of 35–40 °C (optimum, 35 °C). The predominant fatty acids were C16:0 DMA (20.2%), C16:0 (20.2%), C18:0 (10.5%) and C18:1 cis 9 (17.0%). The polar lipids consisted of an unidentified phospholipid, four unidentified glycolipids and three unidentified lipids. Based on its phenotypic analyses, phylogenetic and physiological characteristics, strain BGYT1T represented a novel species within the genus Olsenella, for which the name Olsenella intestinalis sp. nov. is proposed. The type strain is BGYT1T (= KCTC 25379T = GDMCC 1.3011T).


Introduction
The bacterial genus of Olsenella, belonging to the family Atopobiaceae, was first described by Dewhirst et al. (2001). with Olsenella uli as type species. Subsequently, Kraatz et al. (2011) added some phenotypic characteristics to the genus description and described a new species Olsenella umbonata. Genome-based phylogenetic classification of the phylum Actinobacteria showed the genus Olsenella was paraphyletic, but no major emendation for this genus was proposed (Nouioui et al. 2018). More recently, Zgheib et al. (2021) noticed the paraphyletic nature of Olsenella and reclassified the Olsenella species. Subsequently, Lu et al. (2021) transferred Olsenella Communicated by Erko Stackebrandt. gallinarum to genus Thermophilibacter as Thermophilibacter gallinarum. At the end of 2021, the genus Olsenella included seven species with validly published names: O. massiliensis, O. phocaeensis, O. porci, O. profusa, O. uli, O. umbonate and O. urininfantis (https:// lpsn. dsmz. de/ genus/ olsen ella). The genus Olsenella has been found in the healthy bovine rumen, human or pig feces, human gingival crevice, sheep rumen and pig jejunum, respectively (Tajima et al. 2000;Ozutsumi et al. 2005;Cho et al. 2006). The typical characteristics of the genus Olsenella include cells that are anaerobic, Gram stain positive and rod shaped, C 18:1 cis 9 as the major fatty acid, and the G + C content of the DNA is 62-64 mol% (Dewhirst et al. 2001;Göker et al. 2010;Li et al. 2015).
In the present study, strain BGYT1 T was isolated from cow feces during a study investigating the gut microbial diversity of the animal. Polyphasic taxonomic analyses revealed that strain BGYT1 T should be proposed as representing a novel species of the genus Olsenella for which the name Olsenella intestinalis has been proposed.

Isolation, preservation, cultivation conditions
A sample of cow feces used for the isolation of bacteria was collected from the Andong of Republic of Korea. The samples were stored using an anaerobic pouch (GasPakTM EZ) and transported to the laboratory and stored at 4 °C. The isolation and cultivation of bacteria were performed in the anaerobic chamber (Coy Laboratory Products Inc.) filled with 7% CO 2 , 86% N 2 , and 7% H 2 . The sample suspended in phosphate-buffered saline of pH 6.0-8.0 was tenfold serially diluted until 10 -5 , subsequently, 150 μl of suspensions were spread on tryptic soy agar (TSA) supplemented with 5% sheep blood (TSAB). After incubation at 35 °C for 3 days, a large number of colonies were randomly selected, hand-picked with a sterile inoculation loop, subculture and purified, and then incubated at 35 °C for 2 days, a small single colony was selected, designated as BGYT1 T . Cultivated routinely on TSAB at 35 °C, cells were suspended in skim milk (10%, w/v sterilized) stored at − 20 °C for 1 day and then transferred to − 80 °C conditions for long-term preservation. To further analyze the strains closely related to BGYT1 T , two of the closely related phylogenetic relatives to strain BGYT1 T based on 16S rRNA gene sequence comparative analysis were selected and maintained under the same conditions. These reference strains included O. umbonata KCTC 15140 T and O. profusa KCTC 15029 T obtained from the Korean Collection for Type Cultures (KCTC).

Genomic analyses
Genomic DNA was extracted using a PowerSoil ® Pro DNA Isolation Kit (Cat:47014; Qiagen, Carlsbad, CA). The quality of DNA was checked with agarose gel and the integrity and quality were also determined using Qubit (NANODPOP 2000). Sequencing was performed with Illumina Nexa sequencers. Simultaneously, Nanopore was performed for this sample. Briefly, genomic DNA was further sequenced using the MinION platform of Oxford Nanopore Technologies (ONT). Sequencing libraries were prepared using the ligation sequencing kit (SQK-LSK109; ONT) following the manufacturer's protocol (version RPB_9059_v1_revC_08Mar2018) with SPRI bead clean up (AMPure XT beads; Beckman Coulter). Sequencing was performed as multiplex runs on a MinION with Min-Know v1.15.1 using FLO-MIN106 R9.4 flow cells. DNA G + C content of strain BGYT1 T was calculated from the genome data. There were three kinds of methods to calculate genomic correlations between strain BGYT1 T and closely strains of the genus Olsenella contained ANI tool (www. ezbio cloud. net/ tools/ ani), the Genome-to-Genome Page 3 of 8 384 Distance Calculation web server (http:// ggdc. dsmz. de/ distc alc2. php) and AAI calculation tool (http:// enveomics. ce. gatech. edu/) (Meier-Kolthoff et al. 2013;Luo et al. 2014;Yoon et al. 2017b). The genome sequence of strain BGYT1 T was uploaded to the Type Strain Genome Server (TYGS), a free bioinformatics platform for a wholegenome-based taxonomic analysis (https:// tygs. dsmz. de) (Meier-Kolthoff and Göker 2019). The phylogenomic tree was reconstructed using FastME 2.1.6.1 including SPR postprocessing from the genome blast distance phylogeny (GBDP) (Lefort et al. 2015). Branch support was inferred from 100 pseudo-bootstrap replicates each. All genomes including those from the present study were annotated using the same pipeline to annotate to secure the comparison. Prokka was conducted to annotate genomes and generate gff files (Seemann 2014). Functional genes within each genome were also annotated using KEGG and deciphered to pathways using KEGG Decoder (Graham et al. 2018) and KEGG-Expander (https:// github. com/ bjtul ly/ BioDa ta/ tree/ master/ KEGGD ecoder). Rapid Annotation of microbial genomes using Subsystems Technology (RAST) was also used to validate the annotations, particularly subsystems (Overbeek et al. 2014).

Morphology, biochemical and physiologic characteristics
For biochemical and phenotypic analysis, strains were cultivated in an anaerobic chamber for 2 days at 35 °C. The cells were desiccated using a critical point dryer (SPI-Dry Conventional Critical Point Dryer), and were coated with gold by Safematic CCU-010HV high vacuum sputter, subsequently, cell morphology was obtained using a scanning electron microscope. Gram staining was determined under a light microscope using a Gram-stain kit (Difco) according to the manufacturer's instructions. Growth at different pH tolerance (3-9, 1 pH unit interval) was measured by inoculating in pH-adjusted TSB broth and observing the OD value using the microplate reader, the TSB liquid media was adjusted using appropriate biological buffers as a reference. To determine the optimal growth temperature, cells were examined on a TSAB plate for 5 days at various temperatures (15, 20, 25, 30, 35, 40, 45 °C, in 5 °C units) and salt tolerance was estimated by the growing cell in TSAB agar plates with NaCl concentrations ranging from 0.5 to 6%. The aerobic test was measured under aerobic conditions with an anaerobic pouch (GasPakTM EZ) at 35 °C. The catalase test was verified based on bubble formation using a catalase reagent (Bio Mérieux). The oxidase test was examined based on the production of blue color by using an oxidase reagent (Bio Mérieux). Nitrate reduction and indole and urease production were examined using API 20 NE (Bio Mérieux) test. Using API ZYM test strips to determine enzyme activities and other biochemical properties were determined using Rapid ID 32A according to the manufacturer's instructions.
For analysis of polar lipids, compounds were carried out using freeze-dried biomass prepared from cells grown in TSB broth at 35 °C for 4 days. The polar lipids spots were separated by using TLC silica gel 60 F 254 (20 × 20) and spraying with dyes, including 50% H 2 SO 4 , 0.1% ninhydrin (Sigma-Aldrich), molybdenum (Sigma-Aldrich), which used to identify total lipids, amino lipids, and phospholipid, respectively. Cellular fatty acid profiles were determined in cells grown on TSAB agar plate under anaerobic conditions. Strain BGYT1 T is deposited in the Korean Type Culture Collection (KCTC 25379 T ) and the Chinese Microbial Culture Collection (GDMCC 1.3011 T ). The fatty acids were saponified, methylated, and extracted by using the MIDI/ Hewlett Packard Microbial Identification System (Piñeiro-Vidal et al. 2008) based on the manufacturer's processing manuals (Sasser 1990) and subsequently identified using gas chromatography (GC-210; Shimadzu) and Sherlock™ Chromatographic Analysis System software package with the Anaerobic database version 6.1.  Kim et al. (2014). and suggesting that strain BGYT1 T represents a novel species. The phylogenetic trees reconstructed using ML, ME and NJ methods indicated that strain BGYT1 T related to the species in the genus Olsenella and had different locations compared with other Olsenella species (Figs. 1, S1, S2).

Phylogenomics and genomic analyses
The phylogenetic tree based on the TYGS revealed the relationship between strain BGYT1 T and the related type strains (Fig. 2), which showed that strain BGYT1 T was placed in a different species branch from other Olsenella species. Comparison of genomic distances and calculation of the dDDH values between strain BGYT1 T and its closest strains resulted in values below 70%, the cut-off was determined as a threshold for novel species (Meier-Kolthoff et al. 2013).
The ANI values between strain BGYT1 T and its closest relatives reached 76.8%, and calculated ANI values were below the 95-96% threshold for species description, which confirmed the novelty of the species (Ciufo et al. 2018;Jain et al. 2018), respectively. AAI values and dDDH values between strain BGYT1 T and O. umbonata revealed 67.3% and 22.2%, respectively, the obtained values are less than the 95% of AAI and 70% of dDDH threshold for bacterial species classification. (Table 1) (Konstantinidis and Tiedje 2007;Meier-Kolthoff et al. 2013;Luo et al. 2014).
The genome of strain BGYT1 T contained 1835 genes and 8 contigs with a total length of 2,453,694 bp. The N50 value was 604,117 bp. There were fifty tRNAs, six rRNAs (5S, 16S, 23S), and one tmRNA. Based on the wholegenome sequence, the DNA G + C content was 66.9%. In addition, there were one thousand seven hundred and seven-eight CDSs and two BGCs (Table S1). Furthermore, function annotation of the genomes indicated that 62 genes were classified as function unknown of orthologous genes clusters, but a lot of functions known was annotated such as cell wall/membrane/envelope biogenesis, translation, ribosomal structure and biogenesis, carbohydrate transport and metabolism, transcription, signal transduction mechanisms, nucleotide transport and metabolism, defense mechanisms, inorganic ion transport and metabolism, lipid transport and metabolism (Fig. 3). Genes encoding the cell wall degrading enzymes such as chitinases, β-1,3 glucanases, and proteases were also detected in the strain BGYT1 T . Bacteria producing chitinase, glucanase and protease enzymes can be applied to control plant fungal pathogens since chitin, α-and β-glucans and glycoproteins are the major components of the cell walls of fungi (Dimkić et al. 2022).

Taxonomic conclusion
The values for ANI (76.8%), AAI (67.3%), and dDDH (22.2%) compared to the nearest related species were all below the threshold for bacterial species delineation. Some physiological characteristics indicated that strain BGYT1 T was different from these closest strains from the API test. Due to the above information, strain BGYT1 T represents a novel species of the genus Olsenella, which was given the name as Olsenella intestinalis.
Colonies are circular, convex, smooth and entire, 0.2-0.3 mm in diameter on TSAB after incubation at 35 °C for 2 days. Cells are Gram-stain-positive rods with rounded ends, anaerobic, non-motile, cell size in the range 1.5-3.1 μm × 0.2-0.3 μm, oxidase and catalase negative. Growth in pH range 6.0-8.0 and temperature range 35-40 °C with optimum growth at 35 °C and pH 7.0. Cells grow well in presence of 1.5% NaCl, and 2% NaCl inhibits the growth of cells. Positive for alkaline phosphatase, β-glucosidase and arginine dihydeolase. The polar lipids consisted of an unidentified phospholipid, four unidentified glycolipids and three unidentified lipids. The major fatty acids profiles (> 10%) are contained C 16:0 , C 16:0 DMA, C 18:0 and C 18:1 cis 9. The genomic DNA G + C content is 66.9%.
The type strain BGYT1 T (= KCTC 25379 T = GDMCC 1.3011 T ), was isolated from cow feces collected from the Republic of Korea (Andong).