Allobacillus salarius sp. nov., and Allobacillus saliphilus sp. nov., isolated from shrimp paste (ka-pi) in Thailand

Two strains of moderately halophilic, Gram-stain-positive and spore-forming rods, designated as SKP4-8T and SKP8-2T isolated from shrimp paste (Ka-pi), were taxonomic studied based on polyphasic approach. Strain SKP4-8T grew at pH 6.0–9.0 (optimum 7.0), at 25–45 °C (optimum 37 °C) and in 1–16% (w/v) NaCl (optimum 5–10%). Strain SKP8-2T grew at pH 6.0–9.0 (optimum 8.0), at 25–45 °C (optimum 37 °C) and in 0–20% (w/v) NaCl (optimum 3–10%). The strains contained meso-diaminopimelic acid in cell-wall peptidoglycan and the major menaquinone was MK-7. Strain SKP4-8T contained iso-C15:0, anteiso-C15:0 and iso-C17:0; and strain SKP8-2T contained anteiso-C15:0, iso-C15:0, iso-C16:0 and antesio-C17:0 as major cellular fatty acids. Phosphatidylglycerol, diphosphatidylglycerol, unknown phospholipids and an unknown glycolipid were detected as major polar lipids. On the basis of 16S rRNA gene sequence analysis, strains SKP4-8T and SKP8-2T belonged to the genus Allobacillus and were closely related to Allobacillus halotolerans LMG 24826T with 98.8% and 99.3% similarity, respectively. The comparative genome analysis based on average nucleotide identity (ANI) and digital DNA–DNA hybridization revealed that both strains showed the values below 95 and 70%, from each other and from Allobacillus halotolerans LMG 24826T, respectively. Based on the data from this polyphasic study, strains SKP4-8T and SKP8-2T represent novel species of the genus Allobacillus and the name Allobacillus salarius sp. nov. was proposed for SKP4-8T (= KCTC 33905T = LMG 30016T = TISTR 2499T); and Allobacillus saliphilus sp. nov. for SKP8-2T (= KCTC 33906T = LMG 29682T = TISTR 2558T).


Introduction
Shrimp paste (Ka-pi), a dark brown, gray or pink brown in color with strong odor fermented shrimp is commonly used as a cooking ingredient. It is produced by fermentation of shrimp with salt for 1-3 months and has rich in various nutrients, particularly amino acids and peptides, and contains a high concentration of NaCl (Phithakpol et al. 1995;Tanasupawat and Komagata 2001). Moderately halophilic rod-shaped bacteria have been isolated from shrimp paste and was proposed as the new genus Allobacillus (Sheu et al. 2011). In Thailand, many halophilic species were isolated from fermented fish or shrimp paste such as Gracilibacillus thailandensis , Lentibacillus kapialis (Pakdeeto et al. 2007a), Lentibacillus salicampi and Lentibacillus juripiscarius (Namwong et al. 2005), Oceanobacillus kapialis (Namwong et al. 2009), Piscibacillus salipiscarius (Tanasupawat et al. 2007), Salinicoccus siamensis (Pakdeeto et al. 2007b), Virgibacillus kapii (Daroonpunt et al. 2016) and Virgibacillus siamensis ). This study, two strains, SKP4-8 T and SKP8-2 T were isolated from shrimp paste (ka-pi) in Thailand and found to represent the novel species of the genus Allobacillus and were subsequently evaluated by the means of a polyphasic taxonomy.

Phenotypic characteristics
Cells grown on modified JCM medium no. 377 agar at 37 °C after 3 days were examined for their colony and cell morphology. Gram staining was performed as previous described (Barrow and Feltham 1993). Cell form and spore formation were observed under the light microscope (CH-2, Olympus) and electron microscope (JSM-5410LV, Japan). Flagella were examined as described by Forbes (1981). Anaerobic growth was tested by incubating the cultures on modified JCM medium no. 377 plates in an anaerobic jar with AnaeroPack-Anaero (Mitsubishi Gas Chemical, MGC). Catalase and oxidase activities and nitrate reduction were determined as described by Barrow and Feltham (Barrow and Feltham 1993). Hydrolysis of aesculin, gelatin, starch, skim milk and Tween 20, Tween 40, Tween 60 and Tween 80 were determined as described by Namwong et al. (2005). Arginine hydrolysis was performed using a reported medium (Thornley 1960). Acid production from carbohydrates was determined using basal medium composing (g/L) of 5 g carbohydrate, 1 g casamino acids, 1 g yeast extract, 1 g sodium glutamate, 3 g trisodium citrate, 20 g MgSO 4 ·7H 2 O, 2 g KCl, 50 g NaCl, 0.036 g FeCl 2 ·4H 2 O, 0.0036 g MnCl 2 ·4H 2 O and was adjusted to pH 7.0 and incubated at 37 °C for 7 days. Phenol red was used as an indicator. Effects of growth at various NaCl concentrations were investigated in modified JCM medium no. 377 broth omitting MgSO 4 ·7H 2 O with different concentrations of NaCl (0 and 1-20%, w/v with an interval of 1). Growth at different pH (4-10) was investigated in buffered medium (Sorokin 2005) and growth at different temperatures (10-50 °C) was investigated on modified JCM medium no. 377 agar plates. Antibiotic susceptibility was examined by disc diffusion assay (Acar and Goldstein 1991) on Mueller-Hinton agar (Difco) supplemented with 5% (w/v) NaCl. The enzyme activities were determined using API ZYM system (bioMérieux), according to the manufacturer's instructions. All tests were carried out in media supplemented with 5% NaCl, except for the investigation of effects of growth at various NaCl concentrations.

Chemotaxonomy
The cell biomass for chemotaxonomic characterization was produced in modified JCM medium no. 377 at 30 °C for 3 days. Diaminopimelic acid in the cell wall and menaquinone component were determined as described by Komagata and Suzuki (1987). Polar lipids were extracted and analyzed using the procedure of as described by Minnikin et al. 1984 and identified by two-dimensional TLC followed by spraying with appropriate detection reagents (Komagata and Suzuki 1987). For quantitative analysis of cellular fatty acid compositions, cells were cultivated on modified JCM medium no. 377 agar plates at 30 °C for 48 h. Fatty acid methyl esters (FAMEs) were prepared and identified according to the instructions of the Microbial Identification System (MIDI) (Sasser 1990).

Genotypic and phylogenetic analyses
Chromosomal DNA was isolated and purified from cells grown in modified JCM medium no. 377 according to the method of Tamaoka and Komagata (1984). The 16S rRNA gene was amplified, purified, and sequenced as described by Namwong et al. (2005). The sequences were determined by aligning with selected sequences obtained from the Gen-Bank/EMBL/DDBJ database employing CLUSTAL_X version 1.83 (Thompson et al. 1997). The alignment was manually edited and positions with gaps bases were eliminated prior to construction of a phylogenetic tree. The phylogenetic trees based on the neighbor-joining (Saitou and Nei 1987), maximum-likelihood (Felsenstein 1981) and maximum-parsimony methods (Kluge and Farris 1969) were constructed using MEGA version 6 (Tamura et al. 2013).
The confidence values of branches of the phylogenetic tree were determined using bootstrap analyses (Felsenstein 1985) based on 1000 resamplings.
Whole genome sequence of strains SKP4-8 T , SKP8-2 T and Allobacillus halotolerans LMG 24826 T were performed using an Illumina Miseq platform (Illumina, Inc., San Diego, US-CA) by the World Data Center for Microorganisms (WDCM) under the Global Catalogue of Microorganisms (GCM) 2.0 project. Assembling the reads to contigs was accomplished using SPAdes 3.12 (Bankevich et al. 2012). The assembled genome of strains SKP4-8 T , SKP8-2 T and Allobacillus halotolerans LMG 24826 T are publicly available on the GenBank (accession numbers: VMHE00000000, JAGSIE000000000 and JAHLZF000000000, respectively). The genome was annotated using the DFAST server (Tanizawa et al. 2018) following the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). Average nucleotide identity (ANI) values were calculated with pairwise genome alignment of the draft genome sequences of Allobacillus halotolerans LMG 24826 T (JAHLZF000000000) using the ANI-BLAST (ANIb) and ANI-MUMmer (ANIm) algorithms (Meier-Kolthoff and Göker 2019) implemented with the JSpeciesWS web service (Richter and Rosselló-Móra 2009). Calculation of the digital DNA-DNA hybridization (dDDH) values was achieved using the Genome-to-Genome Distance Calculator (GGDC 2.1) using the BLAST + method (Richter et al. 2016). Results were based on the recommended formula 2 (identities/HSP length), which is useful when dealing with incomplete draft genomes.

Results and discussion
Strains SKP4-8 T and SKP8-2 T were Gram-stain-positive, strictly aerobic and spore-forming rods. Endospores are produced at terminal position in swollen sporangia (Supplementary Fig. S1). Colonies were smooth, circular, low convex and cream in the color (1-2 mm in dimeter) after grew on modified JCM medium no. 377 agar at 37 °C for 2 days. Motile by peritrichous flagella (Fig. S2). Strains SKP4-8 T and SKP8-2 T were differentiated from A. halotolerans LMG 24628 T by growth in NaCl, nitrate reduction, aesculin hydrolysis, and acid production from D-fructose, D-galactose, D-glucose and D-mannose (Table 1), and the amount of fatty acids of iso-C 15:0 and anteiso-C 15:0 (Table S1).
The DDBJ accession numbers for the 16S rRNA gene sequence of strains SKP4-8 T is LC215448. The GenBank accession numbers of the draft genome of strain SKP4-8 T is VMHE00000000.
The DDBJ accession numbers for the 16S rRNA gene sequence of strains SKP8-2 T is LC215449. The GenBank accession numbers of the draft genome of strain SKP8-2 T is JAGSIE000000000.
Funding This study was supported by the Grant for International Research Integration: Research Pyramid, Ratchadaphiseksomphot Endowment Fund (GCURP_58_01_33_01), Chulalongkorn University and the International Partnership Program of Chinese Academy of Sciences (Grant No. 153211KYSB 201900211). Additional thanks to the Ratchadapiseksomphot Endowment Fund, Chulalongkorn University for a post-doctoral fellowship to P. K. Table 3 ANIb and ANIm values (%) and the digital DNA-DNA hybridization (dDDH) values between the draft genomes of strains SKP4-8 T , SKP8-2 T and its closely related type strains Draft genomes: 1, Strain SKP4-8 T ; 2, SKP8-2 T ; 3, A. halotolerans LMG 24826 T . All data were determined in this study *Recommended formula (identities/HSP length), which is liberated of genome length and is thus prosperous against the use of incomplete draft genome