DOI: 10.15468/9kvtx8
title: Lactobacillus suantsaii sp. nov., isolated from suan-tsai, a traditional Taiwanese fermented mustard green

description: This dataset contains the digitized treatments in Plazi based on the original journal article Liou, Jong-Shian, Huang, Chien-Hsun, Wang, Chun-Lin, Lee, Ai-Yun, Mori, Koji, Tamura, Tomohiko, Watanabe, Mizuki, Blom, Jochen, Huang, Lina, Watanabe, Koichi (2019): Lactobacillus suantsaii sp. nov., isolated from suan-tsai, a traditional Taiwanese fermented mustard green. International Journal of Systematic and Evolutionary Microbiology 69 (5): 1484-1489, DOI: 10.1099/ijsem.0.003346, URL: http://dx.doi.org/10.1099/ijsem.0.003346AbstractA Gram-stain-positive, rod-shaped, non-motile, catalase-negative and facultative anaerobic strain, L 88 T, was isolated from suan-tsai, a traditional Taiwanese fermented mustard green. Comparative analyses of 16 S rRNA, pheS and rpoA gene sequences demonstrated that strain L 88 T was a member of the genus Lactobacillus. On the basis of 16 S rRNA gene sequence similarity, the type strains of Lactobacillus acidifarinae (98.2 % similarity), Lactobacillus namurensis (98.1 %), Lactobacillus zymae (98.1 %) and Lactobacillus spicheri (96.8 %) were the closest neighbours to this novel strain. The average nucleotide identity, digital DNA--DNA hybridization and average amino acid identity values between L 88 T and its closest relatives were lower than 80, 30 and 90 %, respectively. Phenotypic and genotypic test results demonstrated that strain L 88 T represents a novel species of the genus Lactobacillus, for which the name Lactobacillus suantsaii sp. nov., is proposed. The type strain is L 88 T (= BCRC 12945 T = NBRC 113535 T).The genus Lactobacillus comprises nearly 200 recognized species (www.bacterio.net/lactobacillus.html) at the time of writing, which can be isolated from a wide variety of sources such as fermented foods, fruits, meat, sourdough, vegetables, wine, gastrointestinal and vaginal tracts of humans and animals [1 – 3]. Lactobacilli are Gram-positive, rod-shaped, facultative anaerobic or microaerophilic, non-spore-forming, acid-tolerant, and catalase-negative bacteria with DNA G + C content that is usually less than 50 mol%.The plant-based products of fermented foods play a significant role in human nourishment, such as providing vitamins, minerals, trace elements and other nutrients [4]. Suan-tsai is a spontaneously fermented mustard green (Brassica juncea Coss.) product with a sour flavour that is widely used in traditional foods in Taiwan [5, 6]. It is mainly produced in central Taiwan. The diversity of the lactic acid bacteria (LAB) in suan-tsai has been analysed, and the predominant LAB are Enterococcus faecalis, Lactobacillus alimentarius, Lactobacillus brevis, Lactobacillus coryniformis, Lactobacillus farciminis, Lactobacillus plantarum, Lactobacillus versmoldensis, Leuconostoc citreum, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Pediococcus pentosaceus, Tetragenococcus halophilus, Weissella cibaria and Weissella paramesenteroides [3, 5, 6]. Two novel LAB species (Lactobacillus harbinensis and Lactobacillus futsaii) have been previously isolated from suan-tsai and identified in China and Taiwan, respectively [7, 8]. During a study aimed at the isolation of lactobacilli from different traditional fermented foods, one isolated strain, L 88 T, from suan-tsai was collected in Hsinchu county, Taiwan, and could not be clearly assigned within any recognized species of the genus Lactobacillus by 16 S rRNA gene sequence analysis. Here, we report the phenotypic, chemotaxonomic and genotypic characterization of strain L 88 T.The bacterial genomic DNA was prepared as reported previously [9]. Amplification and sequencing of the 16 S rRNA gene and housekeeping genes (pheS and rpoA) were carried out as previously described [10, 11]. BLAST analysis of all target genes were performed using the EzBioCloud server (www.ezbiocloud.net/taxonomy) [12] and the GenBank database (www.ncbi.nlm.nih.gov/BLAST/). Multiple sequence alignments were performed with the program CLUSTAL_X version 2.1 [13]. The phylogenetic trees were reconstructed using the software package MEGA, version 7.0 [14]. From the novel strain L 88 T and related strains, approximately 1420 bp of the 16 S rRNA gene and approximately 1140 bp of the concatenated two housekeeping gene sequences [pheS (383 bp) and rpoA (755 bp)] were used for reconstructing phylogenetic trees by the maximum-likelihood method with the Jukes–Cantor model [15]. The statistical reliability of the tree was evaluated by bootstrapping analysis of 1000 replicates [16] and the tree topology was also confirmed by the neighbour-joining [17] and minimum-evolution method [18] methods with Kimura’ s twoparameter model. 16 S rRNA gene sequence similarities between strain L 88 T and its closest neighbours, Lactobacillus acidifarinae LMG 22200 T, Lactobacillus namurensis LMG 23584 T, Lactobacillus zymae YIT 12114 T and Lactobacillus spicheri LTH 5753 T, were high, with values of 98.2, 98.1, 98.1 and 96.8 %, respectively. Phylogenetic analysis based on 16 S rRNA gene sequences showed that strain L 88 T belonged to the genus Lactobacillus (Fig. 1). Mattarelli et al. [19] recommended the application of at least two additional phylogenetic markers for accurate identification of the isolates, especially when used for the description of novel species in the genera Bifidobacterium and Lactobacillus. Protein-encoding genes such as pheS and rpoA have been widely applied in phylogenetic analysis of LAB, and showed good resolution with a high discrimination level [6, 8 – 11]. The similarity levels of concatenated housekeeping gene sequences (pheS and rpoA) between strain L 88 T and L. acidifarinae DSM 19394 T, L. namurensis DSM 19117 T, L. zymae DSM 19395 T and L. spicheri DSM 15429 T were 89.8, 88.1, 90.4 and 88.3 %, respectively. The phylogenetic tree based on the concatenated two housekeeping gene sequences reconstructed by the neighbour-joining method showed that the novel strain L 88 T formed an independent cluster which was clearly separated from other closely relatives, and the nodes were supported by high bootstrap values (&gt;90 %). The tree topology was also confirmed by the maximumlikelihood and minimum-evolution methods, indicating that this strain could be represent a novel species within the genus Lactobacillus (Fig. 2).Morphological, cultural and biochemical testing according to standard techniques was performed at 30 Ǫ C, unless otherwise stated. Cell shape and size were observed using phase-contrast microscopy (Eclipse E600, Nikon). Gram staining and catalase and oxidase activities were determined by using cells grown on DeMan, Rogosa and Sharpe (MRS) agar. Standard methods [9] were followed to determine growth at various temperatures and pH, and in the presence of NaCl, the reduction of nitrate and production of ammonia from arginine. Gas production from glucose was measured with a Durham tube in MRS broth. The isomers of lactic acid formed from glucose were determined with a D-/L- lactic acid enzymic kit (R-Biopharm, Roche Diagnostics). Carbohydrate fermentation enzymic activities were determined using the API 50 CH, API 20E and API ZYM systems (bioḾerieux) according to the manufacturer’ s instructions. Fig. 1. Phylogenetic tree based on 16S rRNA gene sequences showing the relationship of Lactobacillus suantsaii sp. nov. L88 T with strains of closely related species. The tree was reconstructed by the maximum-likelihood method on the basis of a comparison of approximately 1419 bp, and Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 T was used as an outgroup. Bootstrap values (&gt;60 %) based on 1000 replicates are shown at branch nodes. Bar, 2 % sequence divergence. Fig. 2. Phylogenetic tree based on the concatenated housekeeping gene sequences (pheS and rpoA) showing the relationship of Lactobacillus suantsaii sp. nov. L88T with strains of closely related species. The tree was reconstructed by the neighbour-joining method on the basis of a comparison of 1138 bp, and Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293T was used as an outgroup. Bootstrap values based on 1000 replicates are shown at branch nodes for neighbour-joining, maximum-likelihood and minimum-evolution methods, respectively. Bar, 5 % sequence divergence. The draft genome was sequenced from an Illumina pairedend library with an average insert size of 350 bp, using an Illumina HiSeq4000 instrument with the PE 150 strategy at the Beijing Novogene Bioinformatics Technology Co., Ltd. (PR China). The low-quality paired-end sequences and adapter sequences were removed with an in-house program. De novo assembly of next-generation sequencing data was performed using SOAPdenovo [20]. The draft genomes were annotated by using the NCBI Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP; www.ncbi.nlm.nih.gov/genomes/static/ Pipeline.html) [21]. A phylogenomic tree was reconstructed as described by Blom et al. [22] using the EDGAR platform. Briefly, the core genome was calculated by implementing a function of EDGAR. Then, multiple sequence alignment of core genes was performed using MUSCLE [23]. Subsequently, the phylogenetic tree was generated using the approximatemaximum-likelihood method by using FastTree 2.0 software [24]. Average nucleotide identity (ANI), digital DNA–DNA hybridization (dDDH) and amino acid identity (AAI) were calculated as described previously [25 – 28]. The draft genome size of strain L 88 T was 2.43 Mb, with G + C content of 51.1 mol%, which contained 2261 total genes, 2172 predicted protein-coding genes, three rRNA operons (5 S rRNA, 16 S rRNA and 23 S rRNA), 63 tRNAs and three noncoding RNAs (ncRNAs) (Table 1). The phylogenomic tree was calculated based on 660 core genes shared by the investigated strains, and showed that strain L 88 T an formed independent cluster (Fig. 3). In addition, the ANI, dDDH and AAI values between the novel strain and closely related strains varied from 75.8--78.4 %, 20.4--22.3 % and 78.3--81.5 %, respectively (Table 2), which were clearly lower than the generally accepted cut-off thresholds (95--96 %, 70 % and 95--96 %) for delineation of prokaryotic species, and thus confirmed that the strain L 88 T represents a novel species.Fig. 1. Phylogenetic tree based on 16S rRNA gene sequences showing the relationship of Lactobacillus suantsaii sp. nov. L88 T with strains of closely related species. The tree was reconstructed by the maximum-likelihood method on the basis of a comparison of approximately 1419 bp, and Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 T was used as an outgroup. Bootstrap values (&gt;60 %) based on 1000 replicates are shown at branch nodes. Bar, 2 % sequence divergence.Fig. 2. Phylogenetic tree based on the concatenated housekeeping gene sequences (pheS and rpoA) showing the relationship of Lactobacillus suantsaii sp. nov. L88T with strains of closely related species. The tree was reconstructed by the neighbour-joining method on the basis of a comparison of 1138 bp, and Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293T was used as an outgroup. Bootstrap values based on 1000 replicates are shown at branch nodes for neighbour-joining, maximum-likelihood and minimum-evolution methods, respectively. Bar, 5 % sequence divergence.The draft genome was sequenced from an Illumina pairedend library with an average insert size of 350 bp, using an Illumina HiSeq4000 instrument with the PE 150 strategy at the Beijing Novogene Bioinformatics Technology Co., Ltd. (PR China). The low-quality paired-end sequences and adapter sequences were removed with an in-house program. De novo assembly of next-generation sequencing data was performed using SOAPdenovo [20]. The draft genomes were annotated by using the NCBI Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP; www.ncbi.nlm.nih.gov/genomes/static/ Pipeline.html) [21]. A phylogenomic tree was reconstructed as described by Blom et al. [22] using the EDGAR platform. Briefly, the core genome was calculated by implementing a function of EDGAR. Then, multiple sequence alignment of core genes was performed using MUSCLE [23]. Subsequently, the phylogenetic tree was generated using the approximatemaximum-likelihood method by using FastTree 2.0 software [24]. Average nucleotide identity (ANI), digital DNA–DNA hybridization (dDDH) and amino acid identity (AAI) were calculated as described previously [25 – 28]. The draft genome size of strain L 88 T was 2.43 Mb, with G + C content of 51.1 mol%, which contained 2261 total genes, 2172 predicted protein-coding genes, three rRNA operons (5 S rRNA, 16 S rRNA and 23 S rRNA), 63 tRNAs and three noncoding RNAs (ncRNAs) (Table 1). The phylogenomic tree was calculated based on 660 core genes shared by the investigated strains, and showed that strain L 88 T an formed independent cluster (Fig. 3). In addition, the ANI, dDDH and AAI values between the novel strain and closely related strains varied from 75.8--78.4 %, 20.4--22.3 % and 78.3--81.5 %, respectively (Table 2), which were clearly lower than the generally accepted cut-off thresholds (95--96 %, 70 % and 95--96 %) for delineation of prokaryotic species, and thus confirmed that the strain L 88 T represents a novel species.Fig. 3. Phylogenomic tree of Lactobacillus suantsaii sp. nov. L88 T with strains of closely related species. The tree was reconstructed by the maximum-likelihood method on the basis of a comparison of 660 core genes. Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 T was used as an outgroup. Bar, 10 % sequence divergence.Table 1. Genomic characteristics of Lactobacillus suantsaii sp. nov.Attribute L88 T Accession no. QXIL00000000 Coverage (×) 410 Genome size (bp) 2 430 870 G+C content (mol%) 51.1 No. of contigs 70 N50 length (bp) 98 901 Total genes 2261 No. of CDS* 2172 RNA genes 75 rRNA Complete 4 Partial 5 tRNA 63 ncRNA a 3 Pseudogenes 89The presence of diaminopimelic acid in the cell-wall peptidoglycan was determined according to Hasegawa et al. [29]. Cell-wall peptidoglycan for analysis of amino acid composition was prepared and hydrolysed as described previously [30]. Cell-wall amino acids were analysed by high-performance liquid chromatography (HPLC) (LC-20AB, Shimadzu) equipped with a Wakopak wakosil-PTC column (200×4.0 mm i.d.; Wako Pure Chemical Industries), as their phenyl isothiocyanate derivatives (Wako). Amino acid isomers in the cell-wall hydrolysate were analysed as described previously [31] using a liquid chromatograph–mass spectrometer (LCMS-2020 and LC-20AB, Shimadzu) equipped with a Shim-Pack FC-ODS column (150×2.0 mm i.d.; Shimadzu). Whole-cell fatty acids were analysed as fatty acid methyl esters with the Sherlock Microbial Identification System (MIDI Inc.) as described previously [32]. The cultures were incubated on MRS agar at 30 Ǫ C for 48 h under anaerobic conditions. Extracts of the methylated fatty acids were prepared according to the protocol provided by the manufacturer and analysed by using an Agilent 6890 N gas chromatograph equipped with a flame ionization detector and a 7683 Automatic Liquid Sampler (Agilent Technologies). Identification of the peaks was made by comparing the results with the built-in TSBA 50 database (MIDI).*CDS, coding sequences (total) based on NCBI PGAAP.†ncRNAs, noncoding RNAs.Various phenotypic features that distinguish the novel species from phylogenetically related species are listed in Table 3. The cellular fatty acid profiles of novel strain and related type strains are listed in Table 4. The novel species could be differentiated from closely related species based on C 20: 0 and summed feature 8 (C 18: 1 Ɯ 7 c and/or C 18: 1 Ɯ 6 c); however, C 20: 0 was a very minor component and summed feature 8 was present in only slightly higher concentrations than in other related species. Strain L 88 T did not contain diaminopimelic acid in the peptidoglycan, instead containing Lys as the diagnostic diamino acid. The peptidoglycan type was determined to A 4a with an interpeptide bridge comprising L- Lys-D- Asp, which was identical to the related type strains. According to the results obtained in the phenotypic characterization, genomic and phylogenetic analysis, and chemotaxonomic analyses, we conclude that the novel strain L 88 T represent a novel species within the genus Lactobacillus, for which the name Lactobacillus suantsaii sp. nov. is proposed, with strain L 88 T (= BCRC 12945 T = NBRC 113535 T) as the type strain.