Lysinibacillus Agricola sp. nov.Isolated from Soil

A Gram-staining-positive, rod-shaped cells, designed strain FJAT-51161 T , was isolated from farmland soil collected from Fujian Province, China. Growth was observed at 25 - 40 °C (optimum 30 °C), pH 7.0-9.0 (optimum 7.0), and NaCl tolerance in the range of 0-7 % (w/v), respectively. Phylogenetic analysis based on the 16S rRNA gene sequences indicated that the strain FJAT-51161 T belonged to the genus Lysinibacillus, and had the closest relationship with Lysinibacillus xylanilyticus XDB9 T (with 99.0 % 16S rRNA sequence similarity). The digital DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) values based on the genome sequence analysis between strain FJAT-51161 T and the closest reference strain were 38.0 % for dDDH and 88.7 % for ANI, respectively, lower than the prokaryotic species dened values. Further analysis showed that strain FJAT-51161 T shared the fatty acid proles such as iso-C 15:0 (46.7%), iso-C 16:0 (15.8%), C 16:1 ω7c alcohol (14.0%), anteiso-C 15:0 (6.9%) with other members of the genus Lysinibacillus. The peptidoglycan was L-Lys–D-Asp (type A4α). The main quinone was MK-7 and MK-6. The major polar lipids were diphosphatidylglycerol (DPG) and phosphatidylethanolamine (PE). The DNA G+C content is 36.6 mol%. Based on the phenotypic characters and taxono-genomics study, strain FJAT-51161 T is considered to represent a novel Lysinibacillus species, for which the name Lysinibacillus agricola sp. nov. is proposed. The type strain is FJAT-51161 T (= GDMCC1.2350 T = KCTC


Introduction
The genus Lysinibacillus was established and transferred from the genus Bacillus by Ahmed et al. in 2007(Ahmed et al. 2007, which belong to the family Bacillaceae of the phylum Firmicutes. The Lysinibacillus species are unique among the family Bacillaceae as they are characterized by a special cell-wall peptidoglycan type of A4α (L-Lys-D-Asp), such as Lysinibacillus yapensis isolated from deep-sea sediment of the Yap Trench, Paci c Ocean (Yu et al. 2019), Lysinibacillus xyleni sp. nov. from a bottle of xylene (Begum et al. 2016), Lysinibacillus louembei sp. nov. from alkaline fermented leaves of cassava (Ouoba et al. 2015), Lysinibacillus manganicus sp. nov. isolated from manganese mining soil (Liu et al. 2013). At the time of writing, the genus Lysinibacillus consisted of 30 species with validly published names (https://lpsn.dsmz.de/genus/lysinibacillus) with Lysinibacillus boronitolerans as the type species (Ahmed et al. 2007). During the survey of Bacillus-like species diversity, an endospore-forming, a novel strain FJAT-51161 T was obtained from soil samples and found to have morphological properties consistent with the genus Lysinibacillus but differentiate from them. Therefore, we adopted polyphasic taxonomic approach combining with the genome indexes to evaluate the taxonomic position of strain FJAT-51161 T .

Materials And Methods
Sample collection, isolation, and preservation Strain FJAT-51161 T was isolated from soil sample of farm land, Fujian Province, China. The sample was serially diluted and an aliquot (100 μL) was spread on LB medium. The plate was incubated at 30 ºC for two days. The colonies obtained were repeatedly re-streaked on the same medium until pure colonies were obtained and stored as glycerol suspensions (20%, w/v) at −80 °C and as lyophilized form in skimmed milk (15 %, w/v) at 4 °C.
Phenotypic, microscopic and growth conditions Colony morphology was observed on LB medium after 24 h of aerobic incubation under optimal growth conditions. The Gram staining and the KOH lysis test were carried out according to the methods described by Gregersen (1978) and Smibert and Krieg (1994). The size of the cells was determined by transmission electron microscopy (Hitachi, Japan). Endospores were examined according to Schaeffer-Fulton staining method (Murray et al. 1994). Motility was examined on motility agar (Chen et al. 2007).

Chemotaxonomy
To investigate chemotaxonomy characters, the peptidoglycan diamino acid test was carried out according to the method described by Schumann (2011). Main quinone was analyzed as described by Collins (1977) using reverse-phase HPLC (Groth et al. 1996). Extraction and analysis of polar lipids by two-dimensional TLC was performed according to Minnikin et al. (1979). The cellular fatty acid pro les of strain FJAT-51161 T and its closely related strains grown on TSBA medium at 28 °C for 24 h were determined according to the Sherlock Microbial Identi cation System (MIDI). The fatty acids were separated using an automated GC system (model 7890N; Agilent) and identi ed with the TSBA6 database of the Microbial Identi cation System (Sasser 1990).

Genome sequencing and comparison
For determination of Digital DDH (dDDH) and Average Nucleotide Identity (ANI), the genome of FJAT-51161 T , L. xylanilyticus DSM 23493 T , L. macroides DSM 54 T and L.contaminans DSM 25560 T were sequenced by the Beijing Novogene Bioinformatics Technology Co., Ltd (China), with accession number CP067341, LFXJ00000000, LGCI00000000 and LGRV00000000. Other genomes were obtained from NCBI database. Estimation of dDDH was performed using the Genome-to-Genome Distance Calculator (GGDC) (Auch et al. 2010;Meier-Kotloff et al. 2013). The genome les were uploaded to the GGDC 2.0 Web interface (http://ggd-c.dsmz.de/ distcalc2.php) and the Formula 2 was used according to the recommendation for the calculation of dDDH for incomplete genomes. The ANI value was calculated using OrthoANIu algorithm (https://www.ezbiocloud.net/tools/ani) according to the description by Yoon et al. (2017b) at the EzGenome web server (http://www.ezbiocloud.net/ezgenome/ani).

Results And Discussion
The colonies of strain FJAT-51161 T were approximate 2 mm in diameter, white-creamy, smooth, opaque circular. The size of cells and presence of agella were determined by transmission electron microscopy (Hitachi, Japan), with a length ranging from 2.0 μm to 3.37 μm and a diameter ranging from 0.8 μm to 1.12 μm ( Supplementary Fig. S1). Growth of strain FJAT-51161 T occurred at 25-40 °C (optimum 30 °C), between 0% and 7.0 % NaCl concentration (optimum 0 %) and pH in the range of 7.0-9.0 (optimum 7.0).
They produced spherical endospores that lay in terminal position. The results showed that strain FJAT-51161 T could not utilize any carbon source to produce acid in API 50 CHB strip. The hydrolysis of gelatin, V-P test and lysine decarboxylase were positive in API 20E, others were negative. The different characteristics of strain FJAT-51161 T in comparison with its closest phylogenetic neighbors were presented in Table 1.
The results of phylogenetic analysis of 16S rRNA gene sequences suggested that strain FJAT-51161 T formed a single branch distinguished from members of the genus Lysinibacillus (Fig. 1). EZBioCloud was obvious that strain FJAT-51161 T should be a member of the genus Lysinibacillus. The phylogenetic position was also con rmed by trees generated using the methods of neighbor-joining ( Supplementary  Fig. 2) and minimum evolution ( Supplementary Fig. 3).
The genome size of FJAT-51161 T was 5, 381, 280 bp, and the genomic DNA G+C content was 36.0 mol%.
Detailed genome features of FJAT-51161 T and closely related members were showed in Table 3. The values of dDDH and ANI for stain FJAT-51161 T with its most closely related species L. xylanilyticus DSM 23493 T were 38.0 % and 88.7 %, respectively, lower than the recognized cut-off values of isDDH >70% and ANI > 95-96% served as a threshold for prokaryotic species delineation (Wayne et al. 1987;Goris et al. 2007;Richter and Rosselló-Móra 2009;Meier-Kolthoff et al. 2013).
Based on the morphological, phenotypic and genotypic distinctiveness (G+C content, 16S rRNA gene sequence and taxono-genomics (dDDH and ANI)), strain FJAT-51161 T can be considered to represent a novel species within the genus Lysinibacillus, for which the name Lysinibacillusagricola sp. nov. is proposed.
Lysinibacillus agricola (a.gri′co.la L. n. ager eld; L. suff. -cola (from L. n. incola) a dweller, inhabitant; L. masc. n. agricola eld dwelling) Cells are aerobic Gram-positive, motile and rod-shaped bacterium with rounded ends. Cells have a length ranging from 2.0 μm to 3.37 μm and a diameter ranging from 0.8 μm to 1.12 μm. Cells are motile by means of lateral agella. On LB plate, the colony diameter is about 1-2 mm, white-creamy, smooth, and opaque. Round endospores are located at terminal position. Growth of strain FJAT-51161 T is achieved aerobically between 25 and 40 °C (optimum 30 °C), between pH 7.0-9.0 (optimum 7.0), and NaCl (w/v) concentration in the range of 0 % to 7.0 % (optimum 0 %). It could not grow at 10% NaCl (w/v). Catalase and oxidase are positive. In API 50CHB strip, strain FJAT-51161 T could not utilize any carbon source to produce acid. In API 20E, hydrolysis of gelatin, Voges-Proskauer test and lysine decarboxylase were positive, others were negative. The main quinone was MK-7. The major polar lipids were diphosphatidylglycerol (DPG) and phosphatidylethanolamine (PE). The peptidoglycan of strain FJAT-51161 T was L-Lys-D-Asp (type A4α). The main quinone was MK-7 and MK-6. The predominant fatty acids are iso-C 15:0 , iso-C 16:0 and C 16:1 ω7c alcohol.
The type strain of the species FJAT-51161 T (=GDMCC1.2350 T = KCTC XXXXX T ) was isolated from soil in Fujian Province, China. The G+C content of the genome is 36.6 mol %.

Author statements
This work was nancially supported by Fujian Academy of Agricultural Sciences (GJPY2019003).

Compliance with ethical standards
Con icts of interest --The authors declared that they had no con ict of interest.