Acetobacter oryzifermentans sp. nov., isolated from Korean traditional vinegar and reclassification of the type strains of Acetobacter pasteurianus subsp. ascendens (Henneberg 1898) and Acetobacter pasteurianus subsp. paradoxus (Frateur 1950) as Acetobacter ascendens sp. nov., comb. nov.

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Acetobacter oryzifermentans sp. nov., isolated from Korean traditional vinegar and reclassification of the type strains of Acetobacter pasteurianus subsp. ascendens (Henneberg 1898) and Acetobacter pasteurianus subsp. paradoxus (Frateur 1950) as Acetobacter ascendens sp. nov., comb. nov. Kyung Hyun Kim a , Ga Youn Cho a , Byung Hee Chun a , Stefan Weckx b , Ji Young Moon c , Soo-Hwan Yeo c,∗∗ , Che Ok Jeon a,∗ a

Department of Life Science, Chung-Ang University, Seoul 06974, Republic of Korea Research Group of Industrial Microbiology and Food Biotechnology, Faculty of Sciences and Bioengineering Sciences, Vrije Universiteit Brussel, B-1050 Brussels, Belgium c Department of Agrofood Resources, National Institute of Agricultural Sciences, RDA, Wanju-gun 55365, Republic of Korea b

a r t i c l e

i n f o

Article history: Received 29 October 2017 Received in revised form 20 March 2018 Accepted 22 March 2018 Keywords: Acetobacter oryzifermentans Acetobacter pasteurianus Acetobacter ascendens New taxa Reclassification Vinegar

a b s t r a c t Twelve Acetobacter pasteurianus-related strains with publicly available genomes in GenBank shared high 16S rRNA gene sequence similarity (>99.59%), but average nucleotide identity (ANI) and in silico DNA-DNA hybridization (DDH) values and multilocus sequence- and genome-based relatedness analyses suggested that they were divided into four different phylogenetic lineages. Relatedness analyses based on multilocus sequences, 1,194 core genes and whole-cell MALDI-TOF profiles supported that strains LMG 1590T and LMG 1591 (previously classified as the type strains of A. pasteurianus subsp. ascendens and paradoxus, respectively) and strain SLV-7T do not belong to A. pasteurianus. Strain SLV-7T , isolated from Korean traditional vinegar, shared low ANI (<91.0%) and in silico DDH (44.2%) values with all other Acetobacter type strains analyzed in this study, indicating that strain SLV-7T represents a new Acetobacter species. The phenotypic and chemotaxonomic analyses confirmed these results and therefore a new species named Acetobacter oryzifermentans sp. nov. is proposed with SLV-7T (= KACC 19301T = JCM 31096T ) as the type strain. Strains LMG 1590T and LMG 1591 shared high ANI (99.4%) and in silico DDH (96.0%) values between them, but shared low ANI (<92.3%) and in silico DDH (<49.0%) values with other type strains analyzed in this study, indicating that strains LMG 1590T and LMG 1591 should be reclassified into a new single species that should be named Acetobacter ascendens sp. nov., comb. nov., with LMD 51.1T (= LMG 1590T = NCCB 51001T ) as its type strain. © 2018 Elsevier GmbH. All rights reserved.

Acetic acid bacteria (AAB) are Gram-negative, obligate aerobic bacteria that mainly oxidize ethanol to acetic acid [59]. Besides acetate production, AAB produce various organic acids, cellulose, surfactants, and pigments, and display interesting physiological and phenotypic properties including biomaterial production, nitrogen fixation, and even animal pathogenicity [45,7,57]. The genus Acetobacter, a representative of AAB, belongs to the family Aceto-

∗ Corresponding author at: Department of Life Science, Chung-An University, 84, HeukSeok-Ro, Dongjak-Gu, Seoul 06974, Republic of Korea. ∗∗ Corresponding author at: Department of Agrofood Resources, National Institute of Agricultural Sciences, RDA, Wanju-gun 55365, Republic of Korea. E-mail addresses: [email protected] (S.-H. Yeo), [email protected] (C.O. Jeon).

bacteraceae within the class Alphaproteobacteria, and at the time of writing, it comprises 48 validated published species and subspecies, which are usually found in sugary, alcoholic, and acidic habitats including several traditional fermented foods and alcoholic beverages, such as vinegar, water kefir, kombucha, sour beers, and in the cocoa bean fermentation process [49,8,60]. Recently, they have been used as starters in the commercial production of vinegars, kombucha, or in cocoa bean fermentation [17,34]. Acetobacter pasteurianus is one of the most well-studied Acetobacter species that has been used to brew diverse vinegars as starter cultures worldwide [17,34]. In the past, A. pasteurianus contained several subspecies named estunensis, lovaniensis, pasteurianus, ascendens, and paradoxus [11], but it was later pro-

https://doi.org/10.1016/j.syapm.2018.03.003 0723-2020/© 2018 Elsevier GmbH. All rights reserved.

Please cite this article in press as: K.H. Kim, et al., Acetobacter oryzifermentans sp. nov., isolated from Korean traditional vinegar and reclassification of the type strains of Acetobacter pasteurianus subsp. ascendens (Henneberg 1898) and Acetobacter pasteurianus subsp. paradoxus (Frateur 1950) as Acetobacter ascendens sp. nov., comb. nov. Syst. Appl. Microbiol. (2018), https://doi.org/10.1016/j.syapm.2018.03.003

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posed that A. pasteurianus subsp. ascendens and A. pasteurianus subsp. paradoxus were heterotypic synonyms of A. pasteurianus [50,18]. In addition, A. pasteurianus subsp. estunensis and A. pasteurianus subsp. lovaniensis were reclassified as new species of the genus Acetobacter, namely as Acetobacter estunensis and Acetobacter lovaniensis, respectively [35]. However, the A. pasteurianus subspecies pasteurianus, ascendens and paradoxus have been used to classify A. pasteurianus strains included in the NCBI taxonomic database, which suggests that a clearer taxonomic description of A. pasteurianus strains is necessary. Sequencing of 16S rRNA genes has been widely used for bacterial identification and classification, but in some cases it is not possible to distinguish between closely related species with high 16S rRNA gene sequence similarities, as is the case for AAB [26,9]. As 16S rRNA gene sequence-based approaches alone are not appropriate to determine the phylogenetic relationships between AAB strains, various other approaches such as 16S-23S rDNA internally transcribed spacer region sequences, (GTG)5 -PCR, multilocus sequence analysis, and whole-cell matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDITOF MS), have been used for this purpose [57,36,2,12,37]. In particular, the introduction of MALDI-TOF MS for the identification and classification of AAB has significantly reduced the time required for analysis. Although these approaches have considerably contributed to progress in the identification and classification of AAB [2,37], they have limited use in the investigation of comprehensive phylogeny to resolve genotypic and phenotypic diversities and differences between AAB, because these methods do not reflect genotypic and phenotypic properties [26,10]. Recently, taxonomic approaches based on the whole bacterial genome facilitated by the development of high-throughput and low-cost sequencing technologies are able to identify and classify bacteria into taxa, more accurately and with higher resolution than the previous traditional taxonomic approaches [23,61]. In this study, we isolated strain SLV-7T , which is very closely related to A. pasteurianus based on 16S rRNA gene sequence similarity, from a Korean traditional vinegar and sequenced its complete genome along with those of A. pasteurianus LMG 1590T and LMG 1591 that were previously classified as the type strains of A. pasteurianus subsp. ascendens and A. pasteurianus subsp. paradoxus, respectively [24]. Here, relatedness analyses based on average nucleotide identity (ANI), and wet-lab and in silico DNADNA hybridization (DDH) values, multilocus sequence analysis (MLSA) of dnaK, groEL, and rpoB genes, core genome-based relatedness, and whole-cell MALDI-TOF MS profiles were performed, and a new species of the genus Acetobacter, Acetobacter oryzifermentans, was proposed. In addition, we proposed to reclassify A. pasteurianus strains LMG 1590 and LMG 1591 as a single novel species, Acetobacter ascendens with LMG 1590T as the type strain. Strain SLV-7T was isolated from a Korean traditional rice vinegar sample. Briefly, 38 Korean traditional rice vinegar samples were collected from all over the Korean peninsula and were serially diluted in phosphate buffered saline (PBS; 150 mM NaCl, 20 mM sodium phosphate, pH 7.0). Aliquots of each serial dilution were spread on YPGD agar (0.5 g yeast extract, 0.5 g peptone, 0.5 g glycerol, 0.5 g d-glucose, and 1.5 g agar per 100 ml) containing 2% (w/v) CaCO3 and 4% (v/v) ethanol. After two days of incubation at 30 ◦ C, a strain, designated SLV-7T , with great acetate-producing ability, was selected. Acetobacter aceti KCTC 12290T (= NCIMB 8621T ), Acetobacter strains KACC 13994T (= LMG 1262T ), LMG 1590, and LMG 1591 (previously classified as the type strains of A. pasteurianus subsp. pasteurianus, A. pasteurianus subsp. ascendens, and A. pasteurianus subsp. paradoxus, respectively), Acetobacter pomorum KCTC 22319T (= LHT 2458T ), and A. pomorum DM001 (a gift from prof. W.J. Lee at Seoul National University) were used as refer-

ence strains for wet-lab DNA–DNA hybridization (DDH) and/or comparison of phenotypic properties, fatty acid compositions and whole-cell MALDI-TOF MS profiles. The 16S rRNA gene of strain SLV-7T was amplified using the F1 (5 - AGAGTTTGATCMTGGCTCAG-3 ) and R13 (5 TACGGYTACCTTGTTACGACTT-3 ) primers and the amplicon was sequenced, as described previously [27]. The obtained sequence was compared with the 16S rRNA gene sequences available in GenBank using the BLAST program (http://www.ncbi.nlm.nih. gov/blast/). The 16S rRNA gene sequences of strain SLV-7T , of the closely related type strains and of closely related strains with the whole genome sequences in GenBank (Table S1) were aligned using the Infernal secondary-structure aware aligner, available within the Ribosomal Database Project [43]. Phylogenetic trees were constructed using the neighbor-joining (NJ), maximumparsimony (MP), and maximum-likelihood (ML) algorithms of the PHYLIP software (version 3.695) [15]. The resulting tree topology was evaluated using bootstrap analysis based on a 1000 times resampled dataset in the PHYLIP package. A MLSA was performed using concatenated nucleotide sequences (7728 bp) of three housekeeping genes [dnaK (1911 bp), groEL (1644 bp), and rpoB (4173 bp)] of Acetobacter strains derived from their whole genome sequences. The concatenated gene sequences were aligned using a web-based tool (CLUSTAL Omega, http://www.ebi.ac.uk/Tools/ msa/clustalo/) [51] and a phylogenetic tree with bootstrap values was constructed using the NJ algorithm in the MEGA7 software [28]. A 16S rRNA gene sequence-based phylogenetic tree including the type strains of all Acetobacter species showed that strain SLV7T was very closely related with strains descirbed in GenBank as A. pasteurianus or A. pomorum, and formed with them a distinct phylogenic lineage from other Acetobacter species (Fig. S1), suggesting that they may have evolved independently from a common ancestor. A phylogenetic tree of A. pasteurianus-related strains using the NJ algorithm showed that strain SLV-7T was clustered with strains DmCS 004 and DM001 (described as A. pomorum in GenBank), with 100% 16S rRNA gene sequence similarities (Fig. 1A ) and the tree topologies generated with the ML and MP algorithms were same (data not shown). However, the latter three strains were not well separated from A. pasteurianus strains LMG 1262T, LMG 1590 and LMG 1591, to which they showed >99.66% 16S rRNA gene sequence similarities. Yet, strains LMG 1590 and LMG 1591, previously known as the type strains of A. pasteurianus subsp. ascendens and subsp. paradoxus, respectively, formed a cluster with strain 3P3, separated from the other A. pasteurianus-related strains. Phylogenetic analysis based on the concatenated sequences of dnaK, groEL, and rpoB genes supported this findings, revealing the same phylogenetic lineages. Recently, it has been suggested that less than 98.65–98.7% 16S rRNA gene sequence similarity can be used as a threshold to avoid laborious DDH for qualifying different species in bacterial classification [52,29,46]. Therefore, wet-lab DDH were performed among A. pomorum KCTC 22319T , strain SLV-7T , and A. pasteurianus strains KACC 13994T , LMG 1590, and LMG 1591, showing more than 98.7% 16S rRNA gene sequence similarities in the phylogenetic tree. DDH experiments were carried out in triplicate using the DIG High Prime DNA labeling kit (Roche Diagnostics GmbH, Mannheim, Germany), according to the procedure described previously [33]. Hybridization signals produced by the hybridization of probes to the homologous target DNA were considered 100%, and signal intensities derived from the hybridizations of serial dilutions were used as a standard for calculating DNA-DNA relatedness. The wet-lab DDH was performed reciprocally (e.g., A × B and B × A). DDH values between strain SLV-7T and the A. pomorum KCTC 22319T and A. pasteurianus strains KACC 13994T , LMG 1590, and LMG 1591 were 35.2 ± 4.5%, 21.3 ± 4.5%, 25.1 ± 10.9%,

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Fig. 1. Neighbor-joining trees based on16S rRNA gene (A) and concatenated dnaK, groEL, and rpoB gene (B) sequences, showing phylogenetic relationships among strain SLV7T , closely related Acetobacter type strains, and closely related Acetobacter strains with whole genome sequences available in GenBank (accession numbers shown between brackets). Strain names proposed newly in this study were used for the tree construction and the type strains were highlighted in bold. Bootstrap values over 70% are shown on the nodes as percentages of 1000 replicates. Acetobacter aceti NBRC 14818T was used as an outgroup. Bars indicate 0.01 changes per nucleotide position.

and 33.4 ± 8.6%, respectively, which were clearly below the 70% threshold of DDH that is generally accepted for delineating different species [53,47]. These results show that strain SLV-7T can be proposed as a new species of the genus Acetobacter. Strains LMG 1590 and LMG 1591 had low DDH relatedness values with the type strains of A. pasteurianus KACC 13994T (37 ± 6.7% and 42 ± 4.2%, respectively), showing that strains LMG 1590 and LMG 1591 are no Acetobacter pasteurianus strains. A high DDH relatedness value was obtained between both strains (90 ± 3.7%), which showed that they belong to the same species. Both strains should also be classified into a single new species of the genus Acetobacter. The whole genome of strain SLV-7 T was completely sequenced. As the whole genomes of A. pasteurianus LMG 1590 and LMG 1591, and A. pomorum (KCTC 22319T ) were not available in GenBank, they were also sequenced. Briefly, the genomic DNA of strains SLV-7T , LMG 1590, LMG 1591, and KCTC 22319T was extracted according to a standard procedure including phenol-chloroform

extraction and ethanol precipitation [54]. The genomes of strains SLV-7T , LMG 1590, and LMG 1591 were completely sequenced using a combination of PacBio RS single-molecule real-time (SMRT) sequencing with a 10-kb library and Illumina Hiseq 2500 sequencing at Macrogen (Seoul, Korea). De novo assembly of sequencing reads derived from the PacBio SMRT sequencing was performed through the hierarchical genome assembly process. Paired-end reads (101 bp) derived from Illumina sequencing were mapped on genome sequences assembled from the PacBio sequencing reads for error corrections. The genome of strain KCTC 22319T was sequenced by an Illumina Hiseq 2500 platform with 101 bp pairedend reads and the high-quality sequencing reads were de novo assembled using the SPAdes software (ver. 3.11.1) [3]. The whole genome sequences of strains SLV-7, LMG 1590, LMG 1591, and KCTC 22319T were deposited in GenBank with accession numbers CP011120-3, CP015164-7, CP015168-71, and PEBQ00000000, respectively.

Please cite this article in press as: K.H. Kim, et al., Acetobacter oryzifermentans sp. nov., isolated from Korean traditional vinegar and reclassification of the type strains of Acetobacter pasteurianus subsp. ascendens (Henneberg 1898) and Acetobacter pasteurianus subsp. paradoxus (Frateur 1950) as Acetobacter ascendens sp. nov., comb. nov. Syst. Appl. Microbiol. (2018), https://doi.org/10.1016/j.syapm.2018.03.003

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Fig. 2. Heat map showing relatedness among Acetobacter pasteurianus and its related strains based on average nucleotide identity (ANI) values (%). Strain names proposed newly in this study are presented as row and column labels and the type strains are highlighted in bold. Squares corresponding to ANI values of less than 95% are colored in blue, showing that the strains are different species. Strains corresponding to red squares with ANI values greater than 95% indicate that theses strains belong to the same species. Color intensities fade as the ANI values approach 95%. Hierarchical clustering on top is represented by a dendrogram, constructed by a simple linkage of ANI values. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

The whole genome sequences of strain SLV-7T , the type strains of A. pasteurianus and A. pomorum, and of A. pasteurianus strains LMG 1590 and LMG 1591 and closely related A. pasteurianus/A. pomorum strains available in GenBank were used to investigate their relatedness based on ANI and in silico DDH. The pair-wise ANI values for whole genomes, including chromosomes and plasmids, were calculated using a stand-alone software available at http://www.ezbiocloud.net/sw/oat [38], with the following recommended parameters: minimum length, 700 bp; minimum identity, 70%; minimum alignment, 50%; BLAST window size, 1000 bp; and step size, 200 bp. The values of in silico DDH among whole genomes were estimated using the server-based genome-to-genome distance calculator ver. 2.1 (http://ggdc.dsmz.de/distcalc2.php) [40], with BLAST+ for genome alignments [4]. The pair-wise relatedness based on ANI and in silico DDH among the A. pasteurianusrelated strains was visualized as heat maps and hierarchical clustering using GENE-E (http://www.broadinstitute.org/cancer/ software/GENE-E/). As the ANI cut-off value has been suggested to be approximately 95–96%, compared to the 70% DDH threshold for prokaryotic species delineation [46,19,48], ANI values based on total genomes among all A. pasteurianus-related strains listed in Table S1 were calculated. The ANI results showed that the A. pasteurianus-related

strains were clustered into four different lineages (Fig. 2). Interestingly, the hierarchical clustering based on ANI values was in relatively good accordance with the topology of the phylogenetic tree based on the 16S rRNA gene sequences. Strain SLV-7T shared high ANI values (98.53–99.88%) with strains DM001 and DmCS 004, but shared less than 91% ANI values with other A. pasteurianus-related strains, indicating that strains SLV-7T , DM001, and DmCS 004 represent a single novel species of the genus Acetobacter. The ANI analysis showed that strains DM001 and DmCS 004, described as A. pomorum in GenBank, shared low ANI values with the type strain of A. pomorum. The type strain of A. pasteurianus LMG 1262T and strains SRCM100623, 386B, NBRC 101655, and NBRC 3283-01 shared relatively high ANI values (97.27–99.40%), showing that they belong to the same species. A. pasteurianus strains LMG 1590, LMG 1591, and 3P3 shared high ANI values (98.30–99.42%) with each other and low ANI values with other A. pasteurianus-related strains, including the A. pasteurianus and A. pomorum type strain, indicating that they should be reclassified as a different species of the genus Acetobacter. Because strain Ab3 also shared low ANI values with all other A. pasteurianus-related strains (90.15–93.56%), strain AB3 can be a new species of the genus Acetobacter. In addition, in silico DDH analysis also showed that the A. pasteurianus-related strains were clustered into four different lin-

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Fig. 3. Heat map showing relatedness among Acetobacter pasteurianus and its related strains based on in silico DNA–DNA hybridization (DDH) values (%). Strain names proposed newly in this study are presented as row and column labels and the type strains are highlighted in bold. Squares corresponding to in silico DDH values of less than 70% are colored in blue, showing that the strains are different species. Strains corresponding to red squares with in silico DDH values greater than 70% indicate that these strains belong to the same species. Color intensity fades as the in silico DDH values approach 70%. Hierarchical clustering on the top is represented by a dendrogram constructed by a simple linkage of in silico DDH values. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

eages, and the hierarchical clustering based on the in silico DDH values was nearly identical to that based on the ANI values (Fig. 3). Strains SLV-7T , DM001, and DmCS 004 shared 40.9–44.2% of in silico DDH values with other A. pasteurianus-related strains, which were little greater than their corresponding wet-lab DDH values (<40%), but these values were clearly lower than the 70% DDH threshold for prokaryotic species delineation, indicating that they are a new species of the genus Acetobacter. The in silico DDH analysis showed that strains DM001 and DmCS 004 had low relatedness with the type strain of A. pomorum like the ANI values, proving that they were misnamed. Strains LMG 1590, LMG 1591, and 3P3 were also clearly distinct from the A. pasteurianus type strain LMG 1262T , with relatively low in silico DDH values (<50%), showing that they were also misnamed. In a previous study [41], 79–80% of in silico DDH values was suggested as a threshold for delineating different microbial subspecies. Strains LMG 1590, LMG 1591, and 3P3 shared 84.2–96% of in silico DDH values, suggesting that strains LMG 1590, LMG 1591, and 3P3 should be classified under a new single species without being divided into subspecies levels. In addition, the in silico DDH values also shows that strain Ab3 represents a new species of the genus Acetobacter. Genome-based phylogenetic analyses were performed using a genome-BLAST distance phylogeny (GBDP) and core gene-based

methods. For a GBDP-based analysis, BLAST+ was used for local alignment and pseudo-bootstrapping with 100 replicates (formula D5, e-value filter 10−8 ) under the greed-with-trimming algorithm was conducted, as described previously [44]. A phylogenetic tree with bootstrap values was inferred using FastME [39]. For a core gene-based analysis, core genes were extracted from the whole genomes of 14 A. pasteurianus-related strains (Table S1) using the USEARCH program within BPGA (ver 1.2), with >90% sequence identity cut-off [5]. The concatenated amino acid sequences of core genes were aligned, and the misaligned regions were removed using the MUSCLE tool [14] within BPGA. A phylogenetic tree with bootstrap values was constructed using the MEGA software (ver. 7) based on the maximum-likelihood algorithm [28]. The genome-based phylogenetic analysis based on a GBDP clearly showed that the A. pasteurianus-related strains were divided into four different phylogenic lineages, which was identical with the phylogenetic tree generated by the concatenated amino acid sequences of 1194 core genes (Fig. 4). The phylogenetic topologies based on the GBDP and core gene-based analyses were fully in accordance with the phylogenetic relatedness based on the concatenated dnaK, groEL, and rpoB gene sequences (Fig. 1B) and ANI and in silico DDH values (Fig. 4), which supports the notion that the A. pasteurianus-related strains have speciated into four differ-

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Fig. 4. Phylogenomic trees of Acetobacter pasteurianus-related strains using whole genome-based GBDP (A) and concatenated amino acid sequences of 1194 core genes (B). Strain names proposed newly in this study are used for the tree construction and the type strains are highlighted in bold. Bootstrap values over 70% are shown on nodes as percentages of 100 replicates. Bars indicate the numbers of substitutions per site. The genome of A. aceti NBRC 14818T was used as an outgroup (not shown).

ent phylogenetic lineages. Strains LMG 1590, LMG 1591, and 3P3 clustered together and separate from the type strain of A. pasteurianus and the other A. pasteurianus-related strains. Strains SLV-7T , DM001, and DmCS 004 formed a phylogenetic lineage distinct from other A. pasteurianus-related strains. Strain Ab3 also formed a distinct lineage from the A. pasteurianus type strain lineage. To investigate relatedness based on the genotypes of the A. pasteurianus-related strains considered, orthologous clustering was performed using the USEARCH program with a 90% sequence identity cut-off. The clustered output was processed to generate a table with gene presence/absence binary matrices in BPGA. The presence/absence of orthologous genes in different genomes was plotted using GENE-E, with one minus the Pearson correlation distances for the clustering of rows (genes) and columns (genomes). Gene gain or loss occurs continuously in the genomes of organisms over time, and generally, closely related strains share more orthologous genes, suggesting that evolutionary relationships among microorganisms can be inferred from the presence/absence of orthologous genes. In total, 6,871 orthologous genes were identified from the genomes of 12 A. pasteurianus-related strains and the type strains of A. pomorum and A. aceti. Heat map analysis of the molecular phenotypes showed that the presence/absence of orthologous genes in A. pasteurianus-related strains was not exactly in accordance with the phylogenetic clusterings generated by the 16S rRNA gene sequences, ANI and in silico

Fig. 5. Heat map and hierarchical clustering of Acetobacter pasteurianus-related strains based on the presence (red) or absence (blue) of orthologous genes. The type strains are highlighted in bold. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

DDH values, and the core genomes lineages shown in Figs. 1–4 (Fig. 5), probably due to some genetic events such as lateral gene transfer. However, the hierarchical clustering generated by Pearson correlation of the molecular phenotypic profiles was generally consistent with those based on the 16S rRNA gene sequences, ANI and in silico DDH values, and core genomes, further supporting the speciation of the A. pasteurianus-related strains into four phylogenetic lineages. Various analyses recommended for the description of new taxa in the genus Acetobacter were performed for the taxonomic characterization of strain SLV-7T and the reclassification of strains LMG 1590 and LMG 1591 [35,30]. The growth of strain SLV-7T in YPGD broth at different temperatures (10, 15, 20, 25, 30, 37, 40, and 45 ◦ C) for 2 days was checked. The growth of SLV-7T at different pH values (3.0–10.0 at 0.5 pH unit intervals) was assessed in YPGD broth at 30 ◦ C for 2 days. YPGD broths with pH values below 5.5, 6.0–7.5, and 8.0–10.0 were prepared using Citrate buffer, Na2 HPO4 -NaH2 PO4 , and Tris-HCl buffers, respectively, as described previously [20] and the pH values were adjusted again if necessary after sterilization (121 ◦ C for 15 min). Cell morphology and motility of strain SLV-7T were investigated using transmission electron microscopy (JEM1010; JEOL, Japan) and phase-contrast microscopy of cells grown on YPGD agar at 30 ◦ C for 2 days. The following properties of strains SLV-7T and DM001, A. aceti KCTC 12290T , A. pomorum KCTC 22319T , A. pasteurianus KACC 13994T , and strains LMG 1590 and LMG 1591 were investigated in parallel under the same conditions. Ethanol tolerance was assessed, as described previously [42]. Briefly, Acetobacter strains were cultured in YPGD broth with shaking at 220 rpm and 30 ◦ C, until OD600 was measured to be 0.8. The cultures were

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K.H. Kim et al. / Systematic and Applied Microbiology xxx (2018) xxx–xxx Table 1 Phenotypic characteristics of A. oryzifermentans sp. nov., A. ascendens sp. nov., comb. nov. and closely related Acetobacter strains. Strains: 1, A. oryzifermentans SLV-7T ; 2, A. oryzifermentans DM001; 3, A. ascendens LMG 1590T ; 4, A. ascendens LMG 1591; 5, A. pasteurianus KACC 13994T (= LMG 1262T ); 6, A. aceti KCTC 12290T (= NCIMB 8621T ); 7, A. pomorum KCTC 22319T (= LHT 2458T ). All analyses were performed in this study. All strains are positive for catalase activity. All strains are negative for the following characteristics: oxidase activity, growth on maltose and methanol, growth in ammonium as the sole nitrogen source, growth on yeast extract with 30% (w/v) d-glucose, and acid production from d-arabinose. Symbols: +, Positive; −, negative. Characteristics

Formation from d-glucose: 5-Keto-d-gluconic acid 2-Keto-d-gluconic acid Growth in 10% (v/v) ethanol Growth on: d-Fructose d-Sorbitol Glycerol Acid production from: l-Arabinose d-Galactose d-Mannose d-Glucose d-Xylose DNA G + C content (mol%)a a

Table 2 Cellular fatty acid compositions (%) of A. oryzifermentans sp. nov., A. ascendens sp. nov., comb. nov., and closely related Acetobacter strains. Taxa; 1, A. oryzifermentans SLV-7T ; 2, A. oryzifermentans DM001; 3, A. ascendens LMG 1590T ; 4, A. ascendens LMG 1591; 5, A. pasteurianus KACC 13994T (= LMG 1262T ); 6, A. aceti KCTC 12290T (= NCIMB 8621T ); 7, A. pomorum KCTC 22319T (= LHT 2458T ). All data were obtained from this study. Data are expressed as percentages for the total fatty acids and fatty acids less than 0.5% in all strains are not indicated. Major components (>5.0%) are highlighted in bold. tr, trace amount (<0.5%). Fatty acid

Strains 1

2

3

4

5

6

7

− − +

− − +

− − +

− − −

− − +

+ + −

− − −

+ − +

+ − +

− + −

− + −

− − −

+ − +

− − −

− − + + + 52.4

+ − + + + 52.4

− − − − − 53.2

− − − − − 53.3

− − − + + 53.1

+ + + + + 57.1

− − − + − 51.7

The G + C contents were calculated from their whole genome sequencing data.

then serially diluted 10-fold in PBS. For the ethanol tolerance tests, 5 ␮l of each dilution was spotted onto YPGD agar containing different concentrations of ethanol. The spotted YPGD agar plates were incubated at 30 ◦ C for 2 days in boxes containing aqueous acetate or ethanol equivalents at test concentrations to prevent ethanol evaporation from the agar plates. Catalase and oxidase activities were tested by the production of oxygen bubbles in 3% (v/v) aqueous hydrogen peroxide solution and the oxidation of 1% (w/v) tetramethyl-p-phenylenediamine (Merck), respectively [55]. The production of 2-keto-d-gluconic acid and 5-keto-d-gluconic acid was determined using the method described by Gosselé et al. [21]. Utilization of ammonium as the sole nitrogen source was tested using Frateur’s modified Hoyer ethanol medium [13]. Growth in 0.5% yeast extract solution containing 30% d-glucose and in basal medium (0.05% yeast extract and 0.3% vitamin-free casamino acids) containing 0.3% of each of the carbon sources, d-fructose, d-sorbitol, glycerol, maltose, or methanol, was tested as described previously [6]. Acid production from l-arabinose, d-arabinose, d-galactose, dmannose, d-glucose, and d-xylose was determined as described by Asai et al. [1]. Cells of strain SLV-7T were Gram-stain-negative and strictly aerobic rods that occurred singly or in groups with the size of 0.7–1.0 ␮m in width and 1.3–1.9 ␮m in length (Fig. S2). The phenotypic features of strains SLV-7T , DM001, LMG 1590, and LMG 1591 are presented in Table 1 and in the species descriptions. Some of the phenotypic features such as catalase and oxidase activities, growth on yeast extract with 30% (w/v) d-glucose, and formation of 5-keto-d-gluconic acid and 2-keto-d-gluconic acid from d-glucose in strains SLV-7T and DM001 and strains LMG 1590T and LMG 1591 were in accordance with the characteristics of members of the genus Acetobacter, whereas other phenotypic features such as growth of strains SLV-7T and DM001 on d-fructose and glycerol and acid production of strains LMG 1590 and LMG 1591 from dmannose, d-glucose, and d-xylose differentiated them from the closely related type strains of the genus Acetobacter (Table 1). Isoprenoid quinones of strain SLV-7T were analyzed using an HPLC (model LC-20A, Shimadzu) system equipped with a reversedphase column (250 × 4.6 mm, Kromasil, Akzo Nobel) and a diode array detector (SPD-M20A, Shimadzu) with methanol-isopropanol (2:1, v/v) as an eluent (1 ml/min), as described previously [31].

7

C14:0 C14:0 2-OH C12:0 aldehyde C16:0 C16:0 2-OH C16:0 3-OH C18:0 C18:0 3-OH C19:0 cyclo ω8c C20:0 Summed feature 8a

Strain 1

2

3

4

5

6

7

4.2 5.7 1.3 10.2 6.6 1.6 2.2 1.4 1.0 tr 64.4

3.5 4.3 – 9.0 5.4 2.8 3.6 1.9 1.8 2.6 62.7

4.3 5.5 – 11.3 4.3 3.0 3.5 1.7 26.1 tr 30.5

4.6 10.3 1.9 9.3 8.1 1.4 2.2 1.2 1.1 tr 58.1

3.6 3.6 0.9 15.5 4.9 0.9 1.3 0.8 13.7 – 52.7

4.5 10.0 1.2 14.5 6.8 0.8 1.7 0.8 0.6 – 57.2

3.0 5.0 1.0 10.6 4.5 1.7 2.6 1.6 3.1 tr 65.9

a Summed feature represents a group of two or three fatty acids that cannot be separated by GLC with the MIDI system. Summed feature 8, C18:1 ω7c and/or C18: ω6c.

The DNA G + C contents of strain SLV-7T and the reference strains were obtained from their complete genome sequences. Strain SLV7T and the reference strains were cultivated in YPGD broth at 30 ◦ C and their microbial cells were harvested at the same growth stage (exponential phase, OD600 = 0.8). The cellular fatty acids of the microbial cells were saponified, methylated, and extracted using the standard MIDI protocol. The fatty acid methyl esters were analyzed by gas chromatography (Hewlett Packard 6890) and identified using the TSBA6 database of the Microbial Identification System (Sherlock ver. 6.0B) as described previously [36,56]. For the whole-cell MALDI-TOF MS analysis, cells were prepared as described in a previous study [25], and MALDI-TOF MS spectra were obtained in four replicates for all test strains using a MALDI-TOF MS Tinkerbell LT instrument (ASTA Inc., Korea), with detection of positive ions in a linear mode. Heat map visualization and hierarchical clustering analysis of the MALDI-TOF MS profiles were performed using heatmap.2 (package gplots) [58] and hclust (package stats) with the ward.D2 agglomerative clustering method, respectively, in the R program [32]. The only respiratory lipoquinone detected in strain SLV-7T was ubiquinone-9 (Q-9), which was in line with all other members of the genus Acetobacter [59,36]. The DNA G + C content of strain SLV-7T was 52.4 mol%, which was placed within the range of other Acetobacter genus strains (Table 1). The major cellular fatty acids (>5% of the total fatty acids) of strain SLV-7T were summed feature 8 (comprising C18:1 ω7c and/or C18:1 ω6c, 64.4%), C16:0 (10.2%), C16:0 2-OH (6.6%), and C14:0 2-OH (5.7%). The overall fatty acid profile of strain SLV-7T was similar to those of other Acetobacter genus strains, but there were some differences in the respective proportions of some fatty acid components (Table 2). Hierarchical clustering based on the protein profiles of seven Acetobacter strains obtained by wholecell MALDI-TOF MS analysis was generally consistent with those based on ANI and in silico DDH values and MLSA- and core genomebased relatedness (Fig. 6). In conclusion, phylogenetic characteristics including ANI and DDH values and core genome-based and genotype-based phylogenetic relatedness showed that strains LMG 1590 and LMG 1591 should be classified as a single new species of the genus Acetobacter. We propose to classify them as A. ascendens sp. nov., comb. nov. Phylogenetic characteristics also showed that the phylogenetic lineage with strain SLV-7T , A. oryzifermentans DM001, and A. oryzifermentans DmCS 004 represent another species of the genus

Please cite this article in press as: K.H. Kim, et al., Acetobacter oryzifermentans sp. nov., isolated from Korean traditional vinegar and reclassification of the type strains of Acetobacter pasteurianus subsp. ascendens (Henneberg 1898) and Acetobacter pasteurianus subsp. paradoxus (Frateur 1950) as Acetobacter ascendens sp. nov., comb. nov. Syst. Appl. Microbiol. (2018), https://doi.org/10.1016/j.syapm.2018.03.003

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isolated from a Korean traditional rice vinegar. The taxonumber is TA00112. Description of A. ascendens (Henneberg 1898) comb. nov. A. ascendens (Henneberg 1898); basonym, Acetobacter pasteurianus subsp. ascendens (Henneberg 1898) and A. pasteurianus subsp. paradoxus (Frateur 1950) The description of this species is as presented by Lisdiyanti et al. [35], Henneberg [22], De Ley and Frateur [11], and Frateur [16] for A. pasteurianus subsp. ascendens (Henneberg 1898) and A. pasteurianus subsp. paradoxus (Frateur 1950) with the following additions. The major fatty acids (>5%) are summed feature 8 (comprising C18:1 ω7c and/or C18:1 ω6c), C16:0 , and C14:0 2-OH. Unable to produce 5-keto-d-gluconic acid and 2-keto-d-gluconic acid from glucose. Growth on medium 10% (v/v) is variable. Does not grow on yeast extract with 30% (w/v) d-glucose. The DNA G + C content of the type strain is 53.2 mol%. Does not grow on d-fructose, glycerol, maltose, and methanol as a sole carbon source, but is able to grow on dsorbitol. Does not use ammonium as a nitrogen source. The type strain is LMD 51.1T (= LMG 1590T = NCCB 51001T ). The taxonumber is TA00113. Acknowledgements

Fig. 6. Heat map profiling and hierarchical clustering of MALDI-TOF MS spectra of eight Acetobacter strains. The MALDI-TOF MS spectra were obtained for strains with four replicates and m/z peaks of the mass range 2000–20,000 m/z were used for hierarchical clustering analysis.

Acetobacter. Therefore, we propose the name a new species Acetobacter oryzifermentans sp. nov. and describe its phenotypic and molecular properties here. However, we do not propose strain Ab3 as a new species, although its ANI and DDH values and core genomebased and genotype-based phylogenic relatedness clearly support the strain as a distinct phylogenetic lineage, because the strain is not available in public culture collection centers (strain Ab3 was patented in China). Description of A. oryzifermentans sp. nov. A. oryzifermentans (o.ry.zi.fer.men’tans. L. n. oryza, rice; N.L. part. adj. fermentans fermenting; N.L. adj. oryzifermentans rice-fermenting) Cells are gram-negative, strictly aerobic, non-motile rods (0.7–1.0 ␮m wide and 1.3–1.9 ␮m long). Colonies on YPGDE agar are beige-colored, circular, convex and smooth after incubation at 30 ◦ C for 2 days. Growth occurs at 15–37 ◦ C (optimum, 30 ◦ C) and pH 3.5–8.0 (optimum, pH 5.5–6.5). Catalase-positive and oxidasenegative. Able to grow in the presence of 10% ethanol. Ethanol is oxidized to acetic acid. Does not produce 2-keto-d-gluconic acid and 5-keto-d-gluconic acid from d-glucose. Unable to grow on yeast extract with 30% d-glucose and medium with ammonium as the sole nitrogen source. Able to grow on d-fructose and glycerol as a sole carbon source, but not on d-sorbitol, maltose, and methanol. Produces acids from d-mannose, d-glucose, and dxylose, but not from d-arabinose and d-galactose. Acid production from l-arabinose is variable. The major ubiquinone is Q-9. The major fatty acids (>5%) are summed feature 8 (comprising C18:1 ω7c and/or C18:1 ω6c), C16:0 , C16:0 2-OH, and C14:0 2-OH. The DNA G + C content of the type strain is 52.4 mol%, based on the whole genome sequence. The type strain is SLV-7T (= KACC 19301T = JCM 31096T ),

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Please cite this article in press as: K.H. Kim, et al., Acetobacter oryzifermentans sp. nov., isolated from Korean traditional vinegar and reclassification of the type strains of Acetobacter pasteurianus subsp. ascendens (Henneberg 1898) and Acetobacter pasteurianus subsp. paradoxus (Frateur 1950) as Acetobacter ascendens sp. nov., comb. nov. Syst. Appl. Microbiol. (2018), https://doi.org/10.1016/j.syapm.2018.03.003