Effusibacillus dendaii sp. nov. isolated from farm soil

A Gram-positive, rod-shaped, spore-forming, thermophilic, and acidophilic bacterium, designated as strain skT53T, was isolated from farm soil in Tokyo, Japan. Under aerobic conditions, the strain grew at 35–55 °C (optimum temperature 44–55 °C) and pH 4.0–6.0 (optimum pH 5.0). Phylogenetic analysis of the 16S rRNA gene sequence showed that the isolate was moderately related to the type strain of Effusibacillus consociatus (94.3% similarity). The G + C content of the genomic DNA was 48.2 mol%, and MK-7 was the predominant respiratory quinone in the strain. The major fatty acids were anteiso-C15:0, iso-C15:0, and iso-C16:0. Based on the phenotypic and chemotaxonomic characteristics, as well as 16S rRNA gene sequence similarity and whole genome analyses, strain skT53T represents a novel species in the genus Effusibacillus, for which the name Effusibacillus dendaii sp. nov. has been proposed. The type strain is skT53T (= NBRC 114101 T = TBRC 11241 T). Supplementary Information The online version contains supplementary material available at 10.1007/s00203-021-02470-9.


Introduction
The family Alicyclobacillaceae was established by da Costa and Rainey (2009) to accommodate only the genus Alicyclobacillus, which includes spore-forming, acidophilic, and thermophilic bacteria with cellular fatty acids comprising ω-alicyclic fatty acids (Wisotzkey et al. 1992). The genera Kyrpidia (Klenk et al. 2011) and Tumebacillus (Steven et al. 2008) have been added to this family since it was first established. Species belonging to the genus Kyrpidia, which contains two species (Bonjour and Arago 1984;Reiner et al. 2018), are spore-forming, thermophilic, and acidophilic; whereas, species belonging to the genus Tumebacillus are spore-forming and mesophilic. Although typical bacteria belonging to the genus Alicyclobacillus possess ω-alicyclic fatty acids, a few members with no detectable ω-alicyclic fatty acids have also been identified; among them, two species named A. consociatus and A. pohliae, which are phylogenetically distinct from previously known species, have been established (Glaeser et al. 2013;Imperio et al. 2008). Based on 16S rRNA gene sequencing analysis, these two species were subsequently reclassified into the new genus Effusibacillus along with E. lacus in 2014 (Watanabe et al. 2014). In the present study, a novel strain, termed skT53 T , isolated from farm soil was taxonomically studied using polyphasic approaches. Herein, we propose a new species of the genus Effusibacillus.
The GenBank/EMBL/DDBJ accession number for the genome sequence of strain skT53 is AP023366. The GenBank/EMBL/ DDBJ accession number for the 16S rRNA gene sequence of strain skT53 is LC586281.
Briefly, 1 g of soil sample was suspended in 4.5 mL of sterile saline, and the suspension was allowed to stand for 10 min prior to the recovery of the supernatant. The supernatant was serially diluted and plated on SKT medium containing (per liter) 0.8 mg Difco nutrient broth, 6 g gellan gum, and 0.16 g CaCl 2 . The inoculated plates were incubated at 37 °C for 4 weeks. To select microorganisms that exhibited growth only in oligotrophic media, colonies formed were transferred to both SKT and LB media plates. Microorganisms that showed growth on SKT medium without demonstrating growth on LB medium were selected. Selected microorganisms were suspended in sterile saline and incubated at 80 °C for 30 min to enable the formation of spores, and each suspension was then plated onto SKT medium. After incubation at 37 °C for 4 weeks, growth of nine microorganisms was observed; these were then collected and stored for further analysis. The nine microorganisms were suspended individually in sterile saline, and the cell suspensions were incubated at 80 °C for 30 min to facilitate spore formation. After incubation, sporulation of all nine microorganisms was confirmed using a Wirtz spore staining kit (Muto Pure Chemicals, Japan). The 16S rRNA genes of the nine microorganisms were amplified through PCR with the primers 27f (= 8f) and 1492r (Turner et al. 1999;Loy et al. 2002), and their sequences were analyzed. Among the nine microorganisms, we selected the one with the lowest similarity in 16S rRNA gene sequences compared to those with known sequences and designated it as strain skT53 T .

Phenotypic and microscopic analysis and growth conditions
Analysis of cell morphology, assessment using Gram staining, determination of catalase and oxidase activities, and examination of optimal medium conditions were performed by the Identification Service of Techno Suruga Lab Co., Ltd., Japan. Cell morphology was observed using a stereomicroscope (SMZ800N; Nikon, Japan). Gram staining results were observed under a light microscope (BX50F4; Olympus, Japan) using a Gram staining kit (Nissui Pharmaceutical, Japan). Catalase and oxidase activities were determined according to the method described by Barrow and Feltham (1993). To assess anaerobic growth, the growth of the strain skT53 on a yeast extract mineral medium plate (DSM Medium 259, https:// www. dsmz. de/ colle ction/ catal ogue/ micro organ isms/ cultu re-techn ology/ list-of-media-for-micro organ isms) was analyzed using the AnaeroPack System (Mitsubishi Gas Chemical, Japan) (Delaney and Onderdonk 1997).
Each experiment for characterization of strain skT53 T was conducted in triplicate at 50 °C (excluding the growth temperature test) using the yeast extract mineral medium with pH adjusted to pH 5.0 (excluding the pH test). To evaluate the optimum growth temperature, cultures were incubated at 11 different temperatures ranging from 30 °C to 80 °C, using liquid media with incubation temperatures ranging from 30 °C to 50 °C as well as solid media with incubation temperatures ranging from 50 °C to 80 °C. To evaluate optimal growth pH, cultures were incubated using solid media with pH adjusted to pH 4.0, 5.0, 6.0, and 7.0, with HCl or NaOH solution according to previously described methods (Sakamoto et al. 2017).
Additionally, yeast extract was tested at a final concentration of 2 g/L. The sugar oxidation test was performed using an API50CH (BioMérieux, France). The enzyme activity test was performed using APIZYM (BioMérieux, France).

Chemotaxonomic analysis
Menaquinone extraction was performed according to the methods described by Collins et al. (1977), and analysis was performed using HPLC (Kroppenstedt 1982). Polar lipids were extracted from 100 mg of freeze-dried cells, purified using the methods described by Minnikin et al. (1979), and analyzed via thin-layer chromatography using chloroform/ methanol/water (65: 25: 4, by volume) in the first direction and chloroform/acetic acid/methanol/water (80: 18: 12: 5, by volume) in the second. Cellular fatty acid methyl esters were identified and quantified by gas chromatography (6890 N; Agilent Technologies, USA) according to the standard protocol of the Sherlock Microbial Identification System (Sasser 1990) with the Sherlock Midi software (version 6.2) and the TSBA6 database. Amino acids of peptidoglycans were analyzed as described previously (Hamada et al. 2012). The isomer of diaminopimelic acid (DAP) in the cell wall peptidoglycan was determined as described by Hasegawa (1983).

The 16S rRNA gene sequencing and phylogenetic analysis
The 16S rRNA gene fragment was amplified using the universal primers 9F and 1510R. The nucleotide sequence of the amplified fragment was determined by Fasmac Co., Ltd. (Japan), using the primers 9F, 515F, 1099F, 536R, 926R, and 1510R (Lane et al. 1985;Turner et al. 1999;Lane 1991;Nakagawa et al. 2001). The almost-complete 16S rRNA gene sequence (1471 nt) of strain skT53 T was compared with that of the type strains of species with valid published names using EzBioCloud (Yoon et al. 2017a). The CLUSTAL X program (Thompson et al. 1997) was used to align the 16S rRNA gene sequence of strain skT53 T with the corresponding sequences of the family Alicyclobacillaceae. Phylogenetic trees were reconstructed using the neighbor-joining (NJ) (Saitou et al. 1987), maximum-likelihood (ML) (Felsenstein 1981), and maximum-parsimony (MP) (Fitch 1971) algorithms using the MEGA X program (Kumar et al. 2018). The resultant tree topologies were evaluated by performing bootstrap analysis (Felsenstein 1985) based on 1000 replicates.

Genome sequencing and analysis
Genomic DNA extraction of the strain skT53 T was conducted using cultured cells and the Wizard® Genomic DNA Purification Kit (Promega, USA). Genome sequencing was performed by Macrogen Japan Co., Ltd. (Japan) using the PacBio RSII. The reads of each strain were assembled using the FALCON-integrated version 2.14. The DNA G + C content of strain skT53 T was 48.2 mol%. The consensus phylogenetic tree was constructed based on the data of a multi-locus alignment of core genes in the strain skT53 T with related species in the NCBI Assembly database using the automated multi-locus species tree (autoMLST) (https:// autom lst. zieme rtlab. com) (Alanjary et al. 2019).
The average nucleotide identity (ANI) and the digital DNA-DNA hybridization (dDDH) values were used to calculate genomic similarities between strain skT53 T and the type strains of other Effusibacillus species. The ANI based on BLAST was determined using the ANI Calculator (https:// www. ezbio cloud. net/ tools/ ani) (Yoon et al. 2017b;Goris et al. 2007), and the dDDH values were calculated using the Genome-to-Genome Distance Calculator 2.1 (GGDC; http:// ggdc. dsmz. de/ distc alc2. php) (Auch et al. 2010;Meier-Kolthoff et al. 2013a, b). Formula 2 was applied to dDDH analysis.

Results and discussion
The cells of strain skT53 T were rod shaped, and Gram staining results indicated that the cells were Gram positive. The diameter of the cells grown on yeast extract mineral medium ranged from 0.6 to 0.8 µm, and their length ranged from 2 to 10 µm. Spores were observed at high temperatures. The phenotypic characteristics of the strain skT53 T are shown in Table 1. anteiso-C 15:0 , iso-C 15:0 , iso-C 16:0 iso-C 14: 0 , iso-C 15: 0 , iso-C 16: 0 iso-C 15: 0 , anteiso-C 15: 0 iso-C 15: 0 , iso-C 16: 0 , iso-C 17: 0 ω-cyclohexane-C 17: 0 , ω-cyclohexane-C 19: 0 iso-C 15: 0 Aerobic growth on: The skT53 T strain did not grow with the sugars tested but grew aerobically on yeast extract, acetate, fumarate, D-lactate and succinate. No sugar was oxidized in the sugar oxidation test using API50CH. The strain showed weak anaerobic growth under the conditions described in the Materials and methods. Evaluation of enzyme activity using APIZYM revealed that the strain skT53 T exhibits alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine allyl amidase, acid phosphatase, and naphthol-AS-BI-phosphohydrase activities.
The 16S rRNA gene sequence indicated that the species most closely related to strain skT53 T were E. consociatus CCUG53762 T strain (94.3%; Glaeser et al. 2013), E. lacus skLN1 T (93.4%; Watanabe et al. 2014) and E. pohliae MP4 (93.5%; Imperio et al. 2008). Strain skT53 T showed less than 91.3% 16S rRNA gene sequence similarity with other members of the family Alicyclobacillaceae. These results are also in line with the classification of strain skT53 T in the genus Effusibacillus. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that strain skT53 T forms a clade with members of the genus Effusibacillus supported by a high bootstrap value (Fig. 1) and ML and MP algorithms (Supplemental Fig. S1 and S2). As shown in Fig. 1 (NJ), strain skT53 T formed a clade with members of the genus Effusibacillus, and the genera Alicyclobacillus, Tumebacillus, and Effusibacillus formed independent clades. In Supplemental Fig. S1 (ML) and S2 (MP), strain skT53 T formed a clade with members of the genera Effusibacillus and Tumebacillus. Members of the genus Tumebacillus formed a daughter clade against the genus Effusibacillus. These results show that strain skT53 T phylogenetically belongs to the genus Effusibacillus. In addition, these results based on the 16S rRNA gene sequences were consistent with the phylogenetic analysis based on the multi-locus alignment of core genes (Fig. 2).
Strain skT53 T exhibited dDDH values of 22.5% for E. lacus DSM 27172 T and 18.8% for E. pohliae DSM 22757 T ; both values are clearly below the 70% threshold for the definition of bacterial species (Wayne et al. 1987). Strain skT53 T exhibited an ANI of 72.0% for E. pohliae DSM 22757 T and 71.8% for E. lacus DSM 27172 T . These results are significantly below 95-96% (Goris et al. 2007) recommended cutoff points for ANI values.
Based on the data presented, we conclude that strain skT53 T represents a novel species of the genus Effusibacillus, for which the name E. dendaii sp. nov. is proposed.
The isolate is a facultatively anaerobic, gram-positive, spore-forming, and rod-shaped bacterium with a cell size of 0.6-0.8 × 2-10 µm. The temperature range for growth is 35-55 °C, and the optimum growth temperature is 44-55 °C. The pH range for growth is 4.0-6.0, and the  Watanabe et al. (2014) and Glaeser et al. (2013). − , Not detected/not reported * Summed features are groups of two or three fatty acids that cannot be separated by GLC using the MIDI system. Summed feature 3 comprises C 16:1 ω7c/C 16:1 ω6c. Summed feature 4 comprises C17:1 iso I / anteiso B  Fig. 2 The consensus phylogenetic tree was built from a multi-locus alignment of core genes in strain skT53 T with Alicyclobacillaceae species in the NCBI Assembly database by using the automated multilocus species tree (autoMLST) (https:// autom lst. zieme rtlab. phosphatidylethanolamine, phosphatidylmethylethanolamine, three unidentified phospholipids, and two unidentified polar lipids. The diagnostic diamino acid in the cell wall peptidoglycan is meso-DAP. The in silico genomic DNA G + C content of the type strain is 48.2 mol%. The type strain, skT53 T (NBRC 114101 T = TBRC 11241 T ), was isolated from farm soil in Adachi-ku, Tokyo, Japan.