Taxonomic revision of the genus Amphritea supported by genomic and in silico chemotaxonomic analyses, and the proposal of Aliamphritea gen. nov.

A Gram-staining-negative, aerobic bacterium, designated strain PT3T was isolated from laboratory-reared larvae of the Japanese sea cucumber Apostichopus japonicus. Phylogenetic analysis based on the 16S rRNA gene nucleotide sequences revealed that PT3T was closely related to Amphritea ceti RA1T (= KCTC 42154T = NBRC 110551T) and Amphritea spongicola MEBiC05461T (= KCCM 42943T = JCM 16668T) both with 98.3% sequence similarity, however, average nucleotide identity (ANI) and in silico DNA-DNA hybridization (in silico DDH) values among these three strains were below 95% and 70%, respectively, confirming the novelty of PT3T. Furthermore, the average amino acid identity (AAI) values of PT3T against other Amphritea species were on the reported genus delineation boundary (64-67%). Multilocus sequence analysis using four protein-coding genes (recA, mreB, rpoA, and topA) further demonstrated that PT3T, Amphritea ceti and Amphritea spongicola formed a monophyletic clade clearly separate from other members of the genus Amphritea. Three strains (PT3T, A. ceti　KCTC 42154T and A. spongicola JCM 16668T) also showed higher similarities in their core genomes compared to those of the other Amphritea spp. Based on the genome-based taxonomic approach, Aliamphritea gen. nov. was proposed together with the reclassification of the genus Amphritea and Aliamphritea ceti comb. nov. (type strain RA1T = KCTC 42154T = NBRC 110551T), Aliamphritea spongicola comb. nov. (type strain MEBiC05461T = KCCM 42943T = JCM 16668T), and Aliamphritea hakodatensis sp. nov. (type strain PT3T = JCM 34607T = KCTC 82591T) were suggested.

Introduction

The genus Amphritea, a member of the family Oceanospirillaceae in the order Oceanospirillales, was first proposed by Gärtner et al. (2008) with the description of Amphritea atlantica, isolated from deep-sea mussels collected from a hydrothermal vent field [1]. Subsequently, six species have been proposed in this genus: Amphritea japonica, and Amphritea balenae from the sediment adjacent to sperm whale carcasses [2], Amphritea ceti from Beluga whale feces [3], Amphritea spongicola from a marine sponge [4], Amphritea opalescens from marine sediments [5] and Amphritea pacifica from a mariculture fishpond [6]. The bacteria in the genus Amphritea are ecophysiologically diverse, and the genus is characterized as rod-shaped, Gram-negative aerobic chemoorganotrophic, motile by means of a single polar flagellum or bi-polar flagella and catalase-positive [1, 4]. Strains in the genus also accumulate poly-β-hydroxybutyrate [1], which has been suggested as contributing to growth gaps in the sea cucumber Apostichopus japonicus [7]. However, no comprehensive studies on genomic characterization of the genus Amphritea have been undertaken.

In the process of collecting reference genomes to understand structure, function and dynamics of sea cucumber microbiome, strain PT3T, phylogenetically unique bacterium affiliated to the genus Amphritea, was isolated from larvae of Apostichopus japonicus. Here, we report the molecular systematics of previously reported Amphritea species and strain PT3T using modern genome-based taxonomic approaches including in silico chemotaxonomy, and propose Aliamphritea gen. nov. with the reclassification of A. ceti and A. spongicola as Aliamphritea ceti comb. nov. and Aliamphritea spongicola comb. nov. and the strain PT3T as Aliamphritea hakodatensis sp. nov.

Materials and methods

Bacterial strains and phenotypic characterization

The strain PT3T was isolated from the pentactula larvae of Apostichopus japonicus reared in a laboratory aquarium in July 2019. Larvae were collected with 45 μm nylon mesh (FALCON Cell Strainer, Durham, USA) and ten-fold serial dilutions of the homogenate were cultured on 1/5 strength ZoBell 2216E agar plates. Bacterial colonies were purified using the same agar plate. A. atlantica JCM 14776T, A. balenae JCM 14781T, A. japonica JCM 14782T, A. spongicola JCM 16668T and A. ceti KCTC 42154T were used as references for genomic and phenotypic comparisons against strain PT3T. All strains were cultured on Marine agar 2216 (BD, Franklin Lakes, New Jersey, USA). The phenotypic characteristics were determined according to previously described methods [8-11].

Cell morphology of the strain PT3T was observed using a transmission electron microscope JEM-1011 (JEOL, Tokyo, Japan). Cells grown in a Marine Broth 2216 (BD) at 25°C for two days were stained with EM Stainer (Nisshin EM Co., Ltd, Tokyo, Japan) on excel-support-film 200 mesh Cu (Nisshin EM Co., Ltd, Tokyo, Japan).

Motility was observed under a microscope using cells suspended in droplets of sterilized 75% artificial seawater (ASW).

Molecular phylogenetic analysis based on 16S rRNA gene nucleotide sequences

The nearly full length 16S rRNA gene sequence (1,404 bp) of strain PT3T was obtained by direct sequencing of PCR-amplified DNA. 27F and 1509R were used as amplification primers, and four primers: 27F, 800F, 920R and 1509R were used for sequencing [12]. The 16S rRNA gene nucleotide sequences of the type strains of the genus Amphritea and other Oceanospirillaceae species were retrieved from RDP (Ribosomal Database Project) [13] and NCBI databases. Sequences were aligned using Silva Incremental Aligner v1.2.11 [14]. A phylogenetic model test and maximum likelihood (ML) tree reconstruction were performed using the MEGAX v.10.1.8 program [15, 16]. ML tree was reconstructed with 1,000 bootstrap replications using Kimura 2-parameter (K2) with gamma distribution (+G) and invariant site (+I) model. In addition, nucleotide similarities among strains were also calculated using the K2 model in MEGAX.

Whole genome sequencing

Genomic DNA of PT3T, A. atlantica JCM 14776T, A. japonica JCM 14782T, A. spongicola JCM 16668T and A. ceti KCTC 42154T was extracted from the cells grown in Marine Broth 2216 using the Wizard genomic DNA purification kit (Promega, Madison, WI, USA) according to the manufacturer’s protocol. Genome sequencing was performed using both Oxford Nanopore Technology (ONT) MinION and Illumina MiSeq platforms. For the ONT sequencing, the library was prepared using Rapid Barcoding Sequence kit SQK-RBK004 (Oxford Nanopore Technologies, Oxford, UK) according to the standard protocol provided by the manufacturer. The library was loaded into flowcell (FLO-MIN 106), and a 48-hour sequencing run with MinKNOW 3.6.0 software was performed. Basecall was performed using Guppy v4.4.1 (Oxford Nanopore Technologies). Genome sequences were also obtained from a 300 bp paired-end library prepared using the NEBNext Ultra II FS DNA Library Prep Kit for Illumina. The ONT and Illumina reads were assembled using Unicycler 0.4.8 [17]. Genomes for A. balenae JCM 14781T, A. opalescens ANRC-JH14T and A. pacifica ZJ14WT were retrieved from the NCBI database, the assembly accession numbers are GCF_014646975.1, GCF_003957515.1 and GCF_016924145.1, respectively [5, 6, 18]. The whole genome sequences were annotated with DDBJ Fast Annotation and Submission Tool (DFAST) [19]. The complete genome sequences acquired in this study were deposited under AP025281-AP025284 and AP025761-AP025762 (bioproject_id PRJDB12633).

Overall genome relatedness indices (OGRIs)

Overall genome relatedness indices (OGRIs) were calculated to determine the novelty of PT3T. Average nucleotide identities (ANIs) calculated using the Orthologous Average Nucleotide Identity Tool (OrthoANI) software [20] using genomes of the PT3T and previously described Amphritea type strains. In silico DDH values were calculated using Genome-to-Genome Distance Calculator (GGDC) 2.1 [21], results based on formula 2 was adopted, being the most robust against incomplete genomes. Average amino acid identities (AAIs) were calculated and compared between PT3T and other related Oceanospirillaceae species (S1 and S2 Tables) using an enveomics toolbox [22].

Multilocus sequence analysis (MLSA)

MLSA was performed as previously described [8, 9]. The sequences of four protein-coding genes (recA, mreB, rpoA, and topA), essential single-copy genes in the taxa examined in this study were obtained from the genome sequences of PT3T, A. ceti KCTC 42154T, A. spongicola JCM 16668T, A. atlantica JCM 14776T, A. japonica JCM 14782T and other related Oceanospirillaceae species (S2 Table, see genome accession number in the description section below). The sequences of each gene were aligned using ClustalX 2.1 [23]. Concatenation of sequences and phylogenetic reconstruction were performed using SplitsTree 4.16.2 [24].

Pan and core genome analysis

A total of eight genomes, including five obtained in this study (PT3T, A. atlantica JCM 14776T, A. balenae JCM 14781T, A. japonica JCM 14782T, A. spongicola JCM 16668T and A. ceti KCTC 42154T) and two retrieved from the NCBI database (A. opalescens ANRC-JH14T and A. pacifica ZJ14WT) were used for pangenome analysis using the program anvi’o v7 [25] based on previous studies [11, 26], with minor modifications. Briefly, contigs databases of each genome were constructed by fasta files (anvi-gen-contigs-database) and decorated with hits from HMM models (anvi-run-hmms). Subsequently functions were annotated for genes in contigs database (anvi-run-ncbi-cogs). KEGG annotation was also performed (anvi-run-kegg-kofams). The storage database was generated (anvi-gen-genomes-storage) using all contigs databases and pangenome analysis was performed (anvi-pan-genome). The results were displayed (anvi-display-pan) and adjusted manually.

Synteny plot

To elucidate intra-species and inter-genus genome synteny among Aliamphritea and Amphritea species, synteny plot analysis was performed using in silico MolecularCloning (In Silico Biology Inc., Yokohama, Japan). A total of five complete genomic sequences determined in this study (PT3T, A. spongicola JCM 1668T, A. ceti KCTC 42154T, A. atlantica JCM 14776T and A. japonica JCM 14782T) were used for this analysis. Plasmid sequences of A. japonica were not used for this analysis.

In silico chemical taxonomy: Prediction of fatty acids, polar lipid and isoprenoid quinone using the comparative genomics approach

The genes encoding key enzymes and proteins for the synthesis of fatty acids (FAs), polar lipids and isoprenoid quinones were retrieved from the genome sequences of PT3T and seven previously described Amphritea species using in silico MolecularCloning ver. 7. Genomic structure and distribution of the genes were compared also using in silico MolecularCloning ver.7. The 3D-structure of FA desaturase encoding genes from some of the strains was predicted using Phyre2 [27].

Results and discussion

Phylogenetic analysis based on 16S rRNA gene nucleotide sequences showed that strain PT3T was affiliated to the members of the genus Amphritea showing 95.4–98.3% sequence similarities, which are below the proposed threshold range for the species boundary, 98.7% [28, 29]. The strain showed high sequence similarities of 98.3% with A. spongicola and A. ceti. The maximum-likelihood tree also revealed that the PT3T formed a monophyletic clade with A. spongicola and A. ceti within the genus Amphritea (Fig 1). Even after the description of A. pacifica [6], two distinct lineages based on 16S gene sequences in the genus Amphritea have never been discussed yet, but the finding of PT3T showed phylogenetically more cohesion of the strain to A. ceti and A. spongicola compared to the other Amphritea species (Fig 1). This observation triggered further assessments of PT3T using molecular phylogenetic network and genomic approaches, which are frequently used in Vibrionaceae taxonomy [11, 30].

Fig 1

A rooted ML tree based on 16S rRNA gene nucleotide sequences of strain PT3T and related type strains.

Numbers shown on branches are bootstrap values (>50%) based on 1,000 replicated analysis from maximum-likelihood algorithm. Bar, 0.1 substitutions per nucleotide position. Sequences trimmed to 1,337 bp were compared (54–1,390 position in Al. ceti RA1T, KJ867528). Escherichia coli K-12 was used as an outgroup.

Genomic features and overall genome relatedness indices (OGRIs)

Comparison of the genomic features of PT3T and the described Amphritea species showed that PT3T, A. spongicola and A. ceti had relatively larger genome sizes (>4.9 Mb) compared to those of other Amphritea species (S1 Table) (see further discussion in “Pan and core genome analysis” section). The ANI values of the PT3T against A. spongicola, A. ceti, A. atlantica, A. pacifica, A. opalescens, A. japonica and A. balenae were 89.0%, 80.1%, 72.2%, 72.2%, 71.3%, 71.7% and 72.3%, respectively (S1 Fig), which are below the species boundary threshold of 95% proposed in previous studies [31]. The in silico DDH values of PT3T against those species were 36.4%, 22.7%, 21.9%, 21.5%, 21.2%, 22.5% and 22.3%, respectively, and these values were also below the species delineation threshold (70%). ANI and in silico DDH confirmed PT3T as a novel species.

PT3T showed relatively high AAI values of 93.7% and 86.9% to A. spongicola and A. ceti (Fig 2), but these values were also below the species delineation boundary, 95–96% [32]. However, the values against other five Amphritea species (A. atlantica, A. balenae, A. japonica, A. opalescens and A. pacifica) were much lower (64.6–67.1%), and these values were on the border line for the genus delineation threshold, 65–66% [9]. The AAI values indicated that PT3T, A. spongicola and A. ceti could affiliate to a novel genus.

Fig 2

AAI matrix using Aliamphritea and related Oceanospirillaceae.

Reference genomes were downloaded from NCBI database (S2 Table).

MLSA network showed that PT3T, together with A. spongicola and A. ceti, form a monophyletic clade distinct from other Amphritea species (Fig 3). MLSA supported the proposal that those three strains should be re-classified separate from any previously described genera on the basis of phylogenetic cohesion.

Fig 3

MLSA network.

A list of strains and their assembly accession is provided in S2 Table.

Phenotypic characterization

PT3T cells observed under a transmission electron microscope were rod-shaped (1.0–1.4 μm in length and 0.5–0.8 μm in diameter) with a single polar flagellum (S2 Fig), these morphological features were consistent with previously reported descriptions of Amphritea [1-6]. PT3T shared several biochemical features with Amphritea species, such as testing positive in oxidase, NaCl requirements for growth, ability to grow at 15°C and 25°C. PT3T could be distinguished from Amphritea spp. by a total of 43 phenotypic and biochemical features (growth at 4, 30, 37 and 40°C, growth in 1 and 10% NaCl, catalase, indole production, hydrolysis of Tween 80, antibiotics susceptibility and 25 carbon assimilation tests) (Table 1). Several traits also distinguished PT3T from the closely related A. spongicola and A. ceti, for example, hydrolysis of Tween 80, utilization of several organic compounds and susceptibility to SXT (Trimethoprim/Sulfamethoxazole), which indicates phenotypic cohesion in PT3T, A. spongicola and A. ceti.

Table 1

Phenotypic characteristics of PT3T and related species from the genus Amphritea.

Characteristics	Al. hakodatensi	Al. ceti	Al. spongicola	A. atlantica	A. japonica	A. balenae	A. opalescens	A. pacifica
OF test	N	N	N	O	N	N	nd	nd
Growth at
4°C	-	-	-	+	+	+	-	-
30°C	+	+	+	+	-	-	+	+
37°C	-	-	-	+	-	-	+	+
40°C	-	-	-	+	-	-	+	-
Growth in NaCl (broth)
0%	-	-	-	-	-	-	+	-
1%	-	+	+	+	+	+	+	+
10%	w	+	-	+	+	+	+	-
Catalase	+	-	w	w	+	-	+	+
Indole production	+	-	+	-	-	-	-	-
Hydrolysis of Tween 80	+	-	+	+	-	+	nd	-
Antibiotic susceptibility
GM120 (Gentamicin 120)	+	+*2	+	+	+ *1	+ *1	nd	nd
GAT5 (Gatifloxacin 5)	+	nt	+	+	nt	nt	nd	nd
CTX30 (Cefotaxime 30)	+	nt	+	+	nt	nt	nd	nd
CB100 (Carbenicillin 100)	+	+*2	+	+	+*2	+*2	nd	nd
CLR15 (Clarithromycin 15)	+	nt	+	+	nt	nt	nd	nd
SXT (Sulfamethoxazole/Trimethoprim)	-	nt	+	+	nt	nt	nd	nd
AM10 (Ampicillin 10)	+	- *2	+	+	+ *1	+ *2	nd	nd
Utilization of
D-Fructose	-	-	-	+	-	-	nd	+
Sucrose	-	-	-	+	-	-	-	nd
D-Gluconate	-	-	-	+	-	-	nd	nd
Succinate	+	+	-	+	+	-	nd	nd
Fumarate	+	-	-	+	+	-	nd	nd
D-Mannitol	-	-	-	-	-	-	-	+
Citrate	+	-	-	+	-	-	+	nd
4-Aminobutanoate	+	-	+	+	-	-	nd	nd
D-Sorbitol	-	-	-	-	-	-	-	+
DL-Malate	+	-	+	+	-	-	nd	nd
D-Glucose	-	-	-	+	-	-	-	+
Acetate	-	-	-	+	-	-	nd	+
5-Aminovalate	-	-	+	+	-	-	nd	nd
Pyruvate	+	+	-	+	-	-	nd	-
L-Proline	+	-	-	+	-	-	nd	+
L-Glutamate	+	-	+	+	-	-	nd	nd
Putrescine	+	-	+	+	+	+	nd	nd
Propionate	-	-	-	+	-	-	nd	nd
D-Raffinose	-	-	-	+	-	-	nd	nd
D-Ribose	-	-	-	+	-	-	nd	nd
DL-Lactate	+	-	+	+	-	-	nd	nd
L-Alanine	+	-	-	+	-	-	nd	nd
L-Asparagine	-	-	-	+	-	-	nd	nd
Glycine	+	-	-	+	-	-	nd	-
L-Histidine	-	-	-	+	-	-	nd	nd
L-Ornithine	-	-	-	+	-	-	nd	nd
L-Serine	+	-	-	+	-	-	nd	-

Amphritea opalescens (Data from [5]); Amphritea pacifica (Data from [6]).

*1: Data from [2]

*2: Data from [3].

Abbreviations: In OF test, O, oxidative; N, no reaction; +, positive; -, negative; w, weakly positive; nt, not tested; nd, no data.

All strains showed growth at 15°C and 25°C and NaCl concentration of 3%, 6% and 8%. All strains tested in this study were positive for oxidase-test and hydrolysis of DNA. All strains tested in this study were negative for hydrolysis of starch, agar and gelatin, utilization of D-mannose, D-galactose, maltose, melibiose, lactose, N-acetylglucosamine, aconitate, meso-erythritol, D-mannitol, glycerol, L-tyrosine, D-sorbitol, α-ketoglutarate, xylose, trehalose, glucuronate, D-glucosamine, cellobiose, amygdalin, arabinose, D-galacturonate, glycerate, L-rhamnose, salicin, L-arginine, and L-citrulline. All strains were susceptible to gentamicin, carbenicillin (100 μg) and clarithromycin (15 μg) but not to sulfamethoxazole/trimethoprim.

Proposal of the novel genus Aliamphritea

The results of the molecular phylogenetic analyses, OGRIs and classical phenotyping showed delineation of A. ceti, A. spongicola and PT3T from other Amphritea species. Here, we propose Aliamphritea gen. nov. with reclassification of Amphritea ceti and Amphritea spongicola as Aliamphritea ceti comb. nov. and Aliamphritea spongicola comb. nov., respectively. The strain PT3T is proposed as Aliamphritea hakodatensis sp. nov., a novel species in the genus Aliamphritea. Phenotypic characteristics of the genus Aliamphritea was compared to other Oceanospirillaceae phenotype results [33, 34], diagnostic feature’s (morphology, flagellar arrangement and growth at 4°C) differences from other genera were found (Table 2). To elucidate uniqueness of genome backbone of the genus Aliamphritea, we further performed genome comparisons, which could also support the proposal of Al. hakodatensis sp. nov.

Table 2

Phenotypic characteristics of Aliamphritea and genus level comparison within the family Oceanospirillaceae.

	Aliamphritea	Amphritea	Marinomonas	Oceanospirillum	Neptuniibacter	Neptunomonas	Nitrincola	Marinobacterium	Pontibacterium
Morphology	Rods or ovoids	Rods	Helical, curved or straight rods	Helical	Rods	Rods	Rods	Rods	Rods
Number and arrangement of flagella	1 polar or none	1 polar or bipolar tufts	1 polar or bipolar tufts	bipolar tufts	nd	1 polar	1 polar	1 polar	1 polar
Optimal temperature (°C)	25–30	20–30	4–40	25–32	15–37	nd	37	37	30
Growth at 4°C	-	+	d	d	-	+	-	+	-
Growth at 45°C	-	-	-	-	-	-	nd	-	-
Optimal NaCl (%) for growth	2.0–2.5	2.0–3.0	nd	0.5–8.0	nd	1.75–7.0	5	0.6–2.9	2.0
Maximal NaCl (%) for growth	8.0	6.0	nd	8.0	6.0	7.0	8.0	11.7	6.0
Nitrate reduction to nitrite	+	+	d	-	-	-	nd	-	+
Oxidase	+	+	d	+	+	+	+	-	-
Catalase	d	+	d	d	+	+	+	+	+
Gelatin liquefaction	-	d	d	d	-	-	-	nd	-
Starch hydrolysis	-	nd	d	-	-	-	-	nd	-
Utilization of
D-Glucose	-	d	+	-	-	+	-	+	+
D-Fructose	-	d	d	-	-	+	-	+	nd
D-Mannose	-	-	d	-	-	-	-	+	-
Sucrose	-	d	d	-	-	-	-	-	nd
Cellobiose	-	-	d	-	-	nd	-	+	nd
D-Mannitol	-	d	d	-	-	+	-	+	-
Glycerol	-	-	d	-	-	+	-	+	nd
Gluconate	-	d	d	nd	-	-	nd	nd	-
L-Arginine	-	-	d	-	+	nd	-	-	-
Acetate	-	d	d	d	+	+	+	+	nd
PHB accumulation	nd	+	-	+	+	+	nd	-	+
Mol% G + C in DNA	47–52	48–51	41–50	45–50	47	46	47.4	54.9	51.5
Major ubiquinone	Q-8	Q-8	Q-8	Q-8	Q-8	Q-8	nd	Q-8	Q-7, Q-8
Type species	Al. ceti	A. atlantica	M. communis	O. linum	N. caesariensis	N. naphthovorans	N. lacisaponensis	M. georgiense	P. granulatum

Original data from [33, 34]. Phenotypic data for Aliamphritea and Amphritea are from this study and [3–6].

+, present in all strains; -, lack in all strains; d, differs among strains; nd, not determined; W, weak reaction.

The pangenome of Amphritea and Aliamphritea species consists of 10,444 gene clusters (33,961 genes) (Fig 4). Genes were classified into Core for the genes present in all strains, Aliamphritea unique for the genes present in Aliamphritea species, and Amphritea unique for the genes present in Amphritea species. Core consisted of 1,660 gene clusters (13,930 genes). COG categories such as J (translation), ribosomal structure and biogenesis and E (amino acid transport) were abundant in this bin. Core also included genes encoding acetyl-CoA acetyltransferase (phaA), acetoacetyl-CoA reductase (phaB) and poly (3-hydroxyalkanoate) polymerase subunit PhaC (phaC), poly (3-hydroxyalkanoate) polymerase subunit PhaE (phaE), completing the pathway from acetyl-CoA to poly-hydroxybutyrate. Genes coding the C4-dicarboxylate TRAP transporter system (DctMPQ), which is responsible for transportation of organic acid such as succinate, fumarate and malate were also present in this bin. Finally, DNase (exodeoxyribonuclease I,III,V,VII) and predicted lipase (phospholipase/carboxylesterase) coding genes were also present in all strains.

Fig 4

Anvi’o representation of the pangenome of the Aliamphritea and Amphritea species.

Layers represent each genome, and the bars represent the occurrence of gene clusters. The darker colored areas of the bars belong to one of the three bins: Core, Aliamphritea unique or Amphritea unique.

Amphritea unique consists of 446 gene clusters (2,326 genes). COG categories such as T (signal transduction mechanisms) and E (amino acid transport system) were abundant in this bin. Amphritea unique also included putative ABC transporter genes yejABEF. The transporter encoded by these genes counteracts antimicrobial peptides (AMPs) produced by animals, possibly contributing to the survival of symbiotic microbes within host environments [35]. As various associations with animal hosts have been discussed in Amphritea, this feature supports possible associations [1, 2, 6].

Aliamphritea unique consists of 1,312 gene clusters (3,999 genes). COG categories such as T (signal transduction mechanisms) and K (transcription) were abundant in this bin. One of the genes which belong to this bin encodes a tyrosine decarboxylase. This enzyme decarboxylates L-tyrosine to produce CO2 and tyramine, which is an important monoamine for invertebrates which plays a similar physiological role to adrenalin for vertebrates. This suggests a possible ecological role of the Aliamphritea species, interacting with the nervous system of host animals through the production of tyramine [36]. In addition to the putative lipase genes distributed in the Core, triacylglycerol lipase genes were also observed in the Aliamphritea unique.

Aliamphritea and Amphritea had different pathways for metabolism of polyamines (S3 Fig). Aliamphritea species had two genes for putrescine biosynthesis, speA and speB. Arginine decarboxylase encoded by speA decarboxylates arginine into agmatine, then agmatine amidonohydrolase encoded by speB produces putrescine through ureohydrolysis of agmatine. speC encodes ornithine decarboxylase, which directly produces putrescine, this gene was only present in Amphritea unique. Putrescin, together with S-adenosylmethioninamine is synthesized into spermidine through putrescine aminopropyltransferase, encoded by speE. Nevertheless, speE is present in both clades, speD, the gene responsible for production of S-adenosylmethioninamine is only present in Amphritea unique. This suggests that only Amphritea species are potentially capable of producing spermidine independently. Finally, genes encoding putrescine—pyruvate aminotransferase (spuC) and 4-guanidinobutyraldehyde dehydrogenase (kauB) were present in both clade species. The two enzymes catalyze the conversion of putrescine to 4-aminobutanoate (GABA) through two step reactions. Putrescine, and GABA are known to be bacteria-derived amines commonly found in gastro-intestinal environment [37]. In particular, GABA is a neurotransmitter widely distributed in animals including sea cucumber [38], which indicates that Aliamphritea species might influence the nervous system of their host during the developmental stage [39]. Finally, putative tricarboxylic transport membrane protein (TctABC) coding genes, which is responsible for the transportation of lactate, pyruvate and citrate were found in all strains except for A. japonica.

Pan-genome analyses also revealed that genes from two COG categories, K (Transcription) and E (Amino acid transport and metabolism functions) were more abundant in Aliamphritea species than those found in Amphritea species, which could contribute to the genome expansion in Aliamphritea species (S1 Table). In particular, number of genes (122 in average) responsible to LysR-type transcription regulators (LTTRs), which occupied 30% of genes categorized in K, was significantly higher (P<0.01, Welch t-test) in Aliamphritea than that (65 in average) in Amphritea. LTTRs are DNA-binding protein transcriptional regulators which are involved in diverse functions including metabolism, quorum sensing, virulence and motility, commonly regulating a single divergently transcribed gene [40]. In-depth genome BLAST comparison did not find in/del of proper gene clusters and/or regions among Aliamphritea and Amphritea genomes, also supporting the idea that the major causes of genome expansion in Aliamphritea were LTTRs. In the aspect of ecophysiology of Aliamphritea spp., they were isolated from feces of Beluga whale, sea sponge, and sea cucumber larvae [3, 4], which are likely to constitute active heterotrophic microbial communities less affected by nutritional limitations. As reported in the Pelagibacter genomes, the rather low number of transcriptional regulators in the genome is related to fewer transcribed protein-coding genes due to nutritional limitations and/or availabilities [41], therefore, fewer-limitations of nutrients for Aliamphritea in their habitat compared to Amphritea spp., when most of them are isolated from cold extreme environments [1, 5], might drive the genome expansions acquiring LTTRs.

Core- and pan-genome analyses with phenotypic features described below also revealed ecological features of Amphritea and Aliamphritea species as byproduct users, utilizing metabolites from another microbe [42]. Genes encoding transporters of organic acids such as citrate, succinate, fumarate, lactate, malate, pyruvate were widely distributed in both clades, suggesting the ecophysiology of strains in those genera. In addition, the absence of major genes responsible for polysaccharide degrading enzymes and the presence of lipase and DNase genes in Amphritea and Aliamphritea genomes suggest effective strategies using lipid and/or nucleic acids surviving in natural assemblages [42]. This is consistent with animal associated ecological features of the Amphritea and Aliamphritea species [1-4], and Al. hakodatensis is also likely to be a member of the Apostichopus japonicus larval microbial consortium.

Possible protease genes maintaining cellular homeostasis such as ATP-dependent protease ClpP were present in the Core bin, but absence of apparent extra-cellular protease genes in the Core also supports the idea that Amphritea and Aliamphritea are byproduct users, who are unlikely to be more efficient consumers of protein and the related peptides [42].

Synteny plot clearly demonstrates similar gene arrangements among Aliamphritea species while showing less similarity to Amphritea species (Fig 5), supporting genomic cohesion of the novel genus. Furthermore, genome comparisons between Al. hakodatensis, A. atlantica and A. japonica (Fig 5C and 5D) indicate an inversion event which likely occurred during the divergence of Aliamphritea and Amphritea. Comparison between the two Amphritea species, A. atlantica and A. japonica also indicated an inversion (Fig 5E). These results implicate multiple genomic inversions, which may be responsible for the divergence of Amphritea and Aliamphritea (S4 Fig).

Fig 5

Syntenic dotplot comparison of Aliamphritea and Amphritea type strains.

Dots closer to the diagonal line represents collinear arrangement between two homologous genes in two genomes. (A), (B) Intra-genus comparison of Aliamphritea species. (C), (D) Inter-genus comparison between Amphritea and Aliamphritea species. (E) Intra-genus comparison of Amphritea species.

In silico chemical taxonomy

Fatty acids, polar lipids and isoprenoid quinones are common subjects for chemotaxonomic analyses. We performed in silico chemotaxonomy among Amphritea and Aliamphritea species based on comparative genomic approach, as an alternative to the more traditional chemotaxonomy (S3 Table).

Reported cellular fatty acids of Amphritea and Aliamphritea species are mainly linear, mono-unsaturated or saturated consisted of C16:0, C16:1 and C18:1, with small amount of C10:0 3-OH (S3 Table) [1-6]. Pangenomic analysis among described Amphritea species reconstructed the basic type II fatty acid biosynthesis (FASII) pathway driven by FabABFDGIVZ and AccABCD, which is very similar to that of E. coli [43] (Fig 6 and Table 3). The FASII pathway could contribute to three major FAs (C16:0, C16:1 and C18:1) of both genera. In particular, long-chain saturated fatty acid (C16:0) is one of the main features of Amphritea and Aliamphritea fatty acids, comprising approximately 10–30% of the total (S3 Table). C16:0 is one of the main products of the FASII pathway, meaning PT3T could produce C16:0 (Fig 6). Mono-unsaturated fatty acids are also major features of the fatty acid profile. C16:1 and C18:1 together make up over 60% of the total fatty acids (S3 Table). Monounsaturated fatty acids can be produced through ω7 mono-unsaturated fatty acid synthesis initiated by isomerization of trans-2-decenoyl-ACP into cis-3-decenoyl-ACP by FabA (Fig 6). After elongation by FabB, the acyl chain is returned to the FASII pathway and goes through further elongation, producing C16:1ω7c and C18:1ω7c [44]. All strains, including Al. hakodatensis strain PT3T　have fabA and fabB, thus it is suggested that this species is also capable of producing C16:1ω7c and C18:1ω7c. Furthermore, 3-hydroxylated FAs, which are the primary fatty acids in lipid A as well as in ornithine-containing lipids, could be supplied by the FASII pathway since 3-hydroxy-acyl-ACP is known to be normally intermediated in the FAS II elongation cycle [44]. Since no genes responsible for the synthesis of ornithine-containing lipids were found in Al. hakodatensis or any other species in either genus, it is likely that 3-hydroxylated FAs in these species originate in lipid A.

Fig 6

Predicted fatty acid synthetic pathway in Amphritea and Aliamphritea species.

Genes encoding each enzyme were present in all strains unless stated otherwise. *1: Only present in Al. hakodatensis, Al. ceti and Al. spongicola. *2: Only present in Al. ceti, A. atlantica, A. japonica, A. balenae and A. pacifica. ACP: acyl-carrier protein; AccABCD: acetyl-CoA carboxylase complex; FabD: malonyl-CoA: ACP transacylase; FabH/FabY: 3-ketoacyl-ACP synthase Ⅲ; FabB: 3-ketoacyl-ACP synthase Ⅰ; FabF: 3-ketoacyl-ACP synthase Ⅱ; FabG: 3-ketoacyl-ACP reductase; FabA: 3-hydroxyacyl-ACP dehydratase/trans-2-decenoyl-ACP isomerase; enoyl-ACP reductase; FabZ: 3-hydroxyacyl-ACP dehydratase.

Table 3

FAS associated genes composition of the eight strains.

	Al. hakodatensis	Al. ceti	Al. spongicola	A. atlantica	A. japonica	A. balenae	A. opalescens	A. pacifica
accA	+	+	+	+	+	+	+	+
accB	+	+	+	+	+	+	+	+
accC	+	+	+	+	+	+	+	+
accD	+	+	+	+	+	+	+	+
fabD	+	+	+	+	+	+	+	+
fabH	+	+	+	-	-	-	-	-
fabY	+	+	+	+	+	+	+	+
fabB	+	+	+	+	+	+	+	+
fabF	+	+	+	+	+	+	+	+
fabG	+	+	+	+	+	+	+	+
fabA	+	+	+	+	+	+	+	+
fabZ	+	+	+	+	+	+	+	+
fabI	-	-	+	+	+	+	-	+
fabV	+	+	+	+	+	+	+	+
Des1	+	+	+	-	-	-	-	-
Des2	+	+	+	-	-	-	-	-
Des3	+	+	+	-	-	-	-	-
Des4	-	-	-	+	-	-	-	+

+: genes presence; -: absence.

Fatty acid desaturase (Des) can produce unsaturated fatty acid as an alternative to the FASII system. While unsaturated fatty acid synthesis by FabA occurs in anaerobic conditions, fatty acid desaturases are known to function in aerobic conditions [45]. Three fatty acid desaturase homologs (Des1-3) were found in the genome of Al. hakodatensis, Al. ceti and Al. spongicola, while only one homolog, Des4 was found in A. japonica and A. balenae (Table 3). 3D-structure prediction using Phyre2 program shows that these enzymes are likely to be stearoyl-CoA desaturase (SCD), with >99.8% confidence score to the template sequence (S4 Table). Amino acid alignment of Des1-4 also reveals the presence of histidine clusters (HXXXH, HXXHH), which are essential for enzyme activity (S5 Fig) [46]. SCD introduces cis double bond at the Δ9 position of palmitoyl-CoA (C16:0) or stearoyl-CoA (C18:0), producing palmitoleoyl-CoA (C16:1 ω7c) or oleoyl-CoA (C18:1 ω9c). While palmitoleic acid (C16:1 ω7c) is ubiquitous in Amphritea cellular fatty acid profiles, there is no record of oleic acid (C18:1 ω9c) in previous chemotaxonomic properties [1-6]. This suggests that Des1-4 is responsible for the production of palmitoleic acid (C16:1 ω7c) in aerobic environments and is unlikely to be involved in synthesis of other unsaturated fatty acids that are unconfirmed in previously described Amphritea and Aliamphritea species, such as polyunsaturated fatty acid (PUFA). In addition to this, PUFA synthase complex consisting of pfa genes and ole genes were not found in any of the eight strains [47].

Branched-chain fatty acid is also unlikely to be produced. Branched-chain fatty acid synthesis requires 3-ketoacyl-ACP synthase Ⅲ which accepts branched chain primers. The Al. hakodatensis strain has the same 3-ketoacyl-ACP synthase Ⅲ genes (fabY, fabH) as Al. spongicola and Al. ceti (Table 3), which have a fatty acid profile which is mostly linear (S3 Table), thus Al. hakodatensis is also unlikely to produce branched-chain fatty acids.

Comparative genome survey of the genes responsible for the FAS II pathway on the Al. hakodatensis genome reveals the presence of a core gene set, which is mostly similar to the other seven strains (Fig 6, Table 3). Synteny and genomic structures of FAS II core genes are likely to be retained between described Amphritea and Aliamphritea species (S6 and S7 Figs), which could lead to the conclusion that the novel strain is capable of producing similar FA profiles, mainly consisting of C16:0, C16:1ω7c, and C18:1ω7c. Al. hakodatensis is also potentially capable of making C10:0 3-OH which is commonly found in both genera because of the presence of the LpxA gene. LpxA is responsible for the incorporation of 3-hydroxyacyl to UDP-N-acetyl-alpha-D-glucosamine, which is a primary reaction to the biosynthesis of lipid A [48].

Pangenomic analysis among Amphritea and Aliamphritea spp. also revealed a complete gene set for the production of PG and PE; plsX, plsY, plsC, cdsA, pssA, psd, pgsA and pgpA (S5 Table). Furthermore, cls, which is responsible for the production of DPG [49], was detected in the genomes of four strains, A. atlantica, A. balenae, A. opalescens and A. pacifica. This suggests that while A. opalescens is the only Amphritea species with DPG in its polar lipid profile (S5 Table) [5], A. atlantica, A. balenae and A. pacifica are also potential DPG producers. Comparative genomics using five complete genomes reveal that Al. hakodatensis possesses gene sets for PG and PE production as polar lipids, showing particularly high similar gene synteny with Al. spongicola and Al. ceti (S8 Fig).

The only respiratory quinone reported from previously described Amphritea and Aliamphritea species is ubiquinone-8 (Q-8) (S3 Table). Biosynthesis of Q-8 consists of nine steps, and Ubi proteins are involved in each reaction (S9 Fig) [50]. The side chain of Q-8 consists of eight isoprene units, originating in the side-chain precursor octaprenyl-diphosphate, which is synthesized by IspAB (S9 Fig). Core genes include ubi genes responsible for the biosynthetic pathway (ubiC, ubiA, ubiD, ubiX, ubiI, ubiG, ubiH, ubiE), and ispAB. In addition to those, core genes include three genes (ubiB, ubiJ, ubiK) coding accessory proteins also required for Q-8 biosynthesis, but with rather hypothetical functions. UbiB is thought to be responsible for the extraction of ubiquinone precursors from the membrane while UbiJ and UbiK is thought to introduce ubiquinone intermediates to Ubi enzymes such as UbiIGHEG [50]. Ubi genes of Al. hakodatensis share a similar gene structure with the other seven strains, showing especially high similarities with other Aliamphritea members (S10 Fig). The distribution of ubiquinone-associated genes of five strains with complete genomes also shows that Al. hakodatensis has similar gene distribution to Al. ceti and Al. spongicola (S11 Fig), which leads to the suggestion that the predominant ubiquinone of Al. hakodatensis is Q-8.

Recently, further development of novel chemotaxonomy tools based on genome information was suggested in the description of bacterial species as an alternative to classical experimental chemotaxonomy [51]. The in silico chemotaxonomy approach is one way to achieve this, and this methodology has been used exclusively in Corynebacterium and Turicella [52]. We also applied the in silico chemotaxonomy approach in this study to Amphritea and Aliamphritea in the class Gammaproteobacteria, to estimate chemotaxonomic features such as fatty acid profiling, respiratory quinone type, and polar lipid profiling. Using complete genome sequences, we can easily predict the backbone of chemotaxonomic features of strains of interest by evaluating the presence/absence of the genes associated with biosynthetic pathways. By comparing these to the E. coli genome, we can find the in silico chemotaxonomy is capable of being applied to bacteria belonging to the class Gammaproteobacteria, which means in silico chemotaxonomy could be used in a wide range of bacterial taxa/species, in which the complete genomes have already been determined. This approach is also effective in predicting the chemotaxonomic features of less common bacterial strains, of which enough bacterial cell mass for chemotaxonomic experiments is unlikely to be collected. However, there are still difficulties in estimating factors regulating fatty acid chain length, and quantitative amounts of fatty acids. Further biochemistry and structural prediction of enzymes and/or proteins responsible to fatty acid synthesis is also needed.

Conclusions

Using the results of modern genome taxonomic study combined with classical phenotyping, which fulfills phylogenetic, genomic, and phenotypic cohesions, we propose Aliamphritea gen. nov. with reclassification of Amphritea ceti RA1T and Amphritea spongicola MEBiC05461T as Aliamphritea ceti comb. nov. (RA1T = KCTC 42154T = NBRC 110551T) and Aliamphritea spongicola comb. nov. (MEBiC05461T = JCM 16668T = KCCM 42943T), respectively. The strain PT3T represents a novel species in the genus Aliamphritea, for which the name Aliamphritea hakodatensis sp. nov. (PT3T = JCM 34607T = KCTC 82591T) is proposed.

Description of Aliamphritea gen. nov

Aliamphritea (A.li. am.phri’tea. L. masc. pron. alinus, other, another; N.L. fem. n. Amphritea, a name of a bacterial genus; N.L. fem. n. Aliamaphritea, the other Amphritea).

Members are mesotrophic, Gram-negative rods or ovoid belonging to the class Gammaproteobacteria. Shows growth at 15°C, 25°C, and 30°C, with 3.0–8.0% (w/v) NaCl. Positive for oxidase. Members of this genus have a typical FAS II pathway gene set. Members also have complete synthetic pathways for the production of phosphatidylglycerol, phosphatidylethanolamine and ubiquinone-8. All members were isolated from marine animals related sources such as Beluga whale feces, marine sponge, and Apostichopus japonicus larvae. DNA G+C content is 47.1–52.2%. The range of estimated genome sizes based on the complete genome sequences is 5.0 Mb to 5.2 Mb. Type species is Aliamphritea ceti.

Description of Aliamphritea ceti comb. Nov

Aliamphritea ceti (ce’ti. L. gen. n. ceti, of a whale). Basonym: Amphritea ceti Kim et al. 2014 [6].

The description is the same as that published for Amphritea ceti by Kim et al. (2014) [6]. The type strain is RA1T = KCTC 42154T = NBRC 110551T. The complete genome nucleotide sequence is deposited in the DDBJ/ENA/GenBank under the accession number AP025282 (PRJDB12633).

Description of Aliamphritea spongicola comb. Nov

Aliamphritea spongicola (spon.gi’co.la. L. fem. n. spongia, a sponge; L. masc./fem. suffix n. -cola (from L. masc./fem. n. incola), inhabitant; N.L. n. spongicola, inhabitant of sponges). Basonym: Amphritea spongicola Jang et al. 2015 [4].

The description is the same as that published for Amphritea spongicola by Jang et al. (2015) [4]. The type strain is MEBiC05461T = KCCM 42943T = JCM 16668T. The complete genome nucleotide sequence is deposited in the DDBJ/ENA/GenBank under the accession number AP025283 (PRJDB12633).

Description of Aliamphritea hakodatensis sp. Nov

Aliamphritea hakodatensis sp. nov. (ha.ko.da.ten’sis. N.L. fem. adj. hakodatensis, from Hakodate, referring to the isolation site of the strain).

Gram-negative, motile with single polar flagellum. Cells are rod-shaped, 1.0–1.4 μm in length and 0.5–0.8 μm in diameter. Colonies on MA are cream and 0.5–0.75 mm in diameter after culture for 3 days. No pigmentation and bioluminescence are observed. The DNA G+C content is 52.2% and genome size is 5.21 Mb. Growth occurs at 15°C, 25°C and 30°C, with NaCl concentration of 3%, 6%, 8%, 10%. Susceptible for ampicillin (10 μg), cefotaxime (30 μg), gatifloxacin (5 μg), Positive for oxidase- and catalase-test, production of indole, nitrate reduction, hydrolysis of tween 80 and DNA, utilization of succinate, fumarate, citrate, g-aminobutyrate, DL-malate, pyruvate, L-proline, L-glutamate, putrescine, DL-lactate, L-alanine, glycine and L-serine. Negative for hydrolysis of starch, agar and gelatin, utilization of D-mannose, D-galactose, D-fructose, sucrose, maltose, melibiose, lactose, D-gluconate, N-acetylglucosamine, aconitate, meso-erythritol, D-mannitol, glycerol, L-tyrosine, D-sorbitol, α-ketoglutarate, xylose, D-glucose, trehalose, glucuronate, acetate, D-glucosamine, δ-aminovalate, cellobiose, propionate, amygdalin, arabinose, D-galacturonate, glycerate, D-raffinose, L-rhamnose, D-ribose, salicin, L-arginine, L-asparagine, L-citrulline, L-histidine and L-ornithine.

The type strain PT3T (JCM 34607T = KCTC 82591T) was isolated from a pentactula larvae of Apostichopus japonicus reared in a laboratory aquarium in Hokkaido University, Hakodate, Japan. The GenBank accession number for the 16S rRNA gene sequence of the type strain is OL455018. The complete genome sequence of the strain is deposited in the DDBJ/ENA/GenBank under the accession number AP025281 (PRJDB12633).

Heat map representation of ANI values of Aliamphritea and Amphritea species.

(PDF)

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An electron micrograph of negatively stained Aliamphritea hakodatensis PT3T cell.

The bar represents 1 μm.

(PDF)

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Predicted polyamine metabolism pathways in Aliamphritea and Amphritea species.

(PDF)

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Evolutionary history of Aliamphritea and Amphritea genome arrangement.

(PDF)

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Amino acid sequence alignment of Des1-4.

(PDF)

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Genomic distribution of fab and associated genes.

Protein/enzyme name each gene is coding: fabA: 3-hydroxyacyl-ACP dehydrase/trans-2-decenoyl-ACP isomerase; fabB: 3-ketoacyl-ACP synthase Ⅰ; fabD: malonyl-CoA: ACP transacylase; fabF: 3-ketoacyl-ACP synthase Ⅱ; fabG: 3-ketoacyl-ACP reductase; fabH: 3-ketoacyl-ACP synthase Ⅲ; fabI: enoyl-ACP reductase Ⅰ; acetyl-CoA; fabV enoyl-ACP reductase; fabY: 3-ketoacyl-ACP synthase; fabZ: 3-hydroxyacyl-ACP dehydratase; accABCD: carboxylase complex; plsX: phosphate acyltransferase; acpP: Acyl-carrier protein.

(TIF)

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Genomic structure of Amphritea and Aliamphritea fab and associated genes.

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Genomic distribution of genes associated with PG, PE and DPG production.

plsC: 1-acyl-sn-glycerol-3-phosphate acyltransferase; plsX: phosphate acyltransferase; plsY: acyl phosphate: glycerol-3-phosphate acyltransferase cdsA: phosphatidate cytidylyltransferase; pssA: CDP-diacylglycerol—serine O-phosphatidyl transferase; psd: phosphatidylserine decarboxylase; pgsA: CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase; pgpA: phosphatidyl glycerophosphatase A; cls: cardiolipin synthase.

(TIF)

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Predicted Q-8 synthetic pathway in Amphritea and Aliamphritea species.

(PDF)

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Genomic structure of ubi and associated genes.

(PDF)

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Genomic distribution of ubiquinone associated genes.

ubiA: 4-hydroxybenzoate polyprenyltransferase; ubiB: protein kinase; ubiC: chorismite lyase; ubiD: 4-hydroxy-3-polyprenylbenzoate decarboxylase; ubiE: dimethylmenaquinone methyltransferase / 2-methoxy-6-polyprenyl-1,4-benzoquinol methylase; ubiF: 3-demethoxyubiquinol 3-hydroxylase; ubiG: 2-polyprenyl-6-hydroxyphenyl methylase / 3-demethylubiquinone-9 3-methyltransferase; ubiH: 2-octaprenyl-6-methoxyphenol hydroxylase; ubiI: 2-polyprenylphenol 6-hydroxylase; ubiJ: ubiquinone biosynthesis accessory factor; ubiK: ubiquinone biosynthesis accessory factor; ubiX: flavin prenyltransferase; ispA: farnesyl diphosphate synthase; ispB: octaprenyl-diphosphate synthase.

(TIF)

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Genome properties of Aliamphritea and Amphritea species.

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List of other Oceanospirillaceae genomes used for genome taxonomy of Aliamphritea and Amphritea.

+: used, -: not used.

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Fatty acid, isoprenoid quinone and polar lipid profile of previously reported Amphritea and Aliamphritea species.

PG, phosphatidylglycerol; PE, phosphatidylethanolamine; DPG, diphosphatidylglycerol; GPL: glycophospholipid. nd: not determined.

(PDF)

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Results of 3D-structure prediction of Des1-4 by Phyre2.

(PDF)

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PG, PE and DPG associated genes composition of each strain.

(PDF)

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15 Feb 2022

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The electronic version of this article in Portable Document Format (PDF) in a work with an ISSN or ISBN will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants, and hence the new names contained in the electronic publication of a PLOS ONE article are effectively published under that Code from the electronic edition alone, so there is no longer any need to provide printed copies.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Major comments

The paper by Yamano et al describes a microbial isolate and reclassfication as a new genus and new species of the family Oceanospirillaceae. Classification using the genome as a definition of a new genus is good. However, since there is no chemical classification information, it is necessary to analyze the bacterial cell fatty acids of the isolates and compare them with Aliamphritea ceti and Aliamphritea spongicola.

Minor comments

L36: “sp., nov.” -> “sp. nov.”

L80: What does the abbreviation “ASW” mean?

L95: Delete this word "(BD)"

L111: What does the abbreviation “DDH” mean?

L119: These gene (recA, mreB, rpoA, and topA) different from the abstract is described.

L141: JCM1668T -> JCM 16668T

L163: JAMM 1525T -> JCM 14781T

L178: Please change the following reference "(Lee et al., 2018)" to a number.

L184: The minimum and maximum values in Fig. 2 are different.

L217-238: I don't know where the explanations in Fig6, Fig.7, and Table2 are separated.

Reviewer #2: The authors described novel genus by dividing the genus Amphritea based on genomic classification criteria and suggested a novel species in the novel genus. The suggestions looks reliable but required something more for demonstration. Sometimes English grammar has problems. Details are as follows.

1. About the Title and basic suggestion

A. The type species of the novel genus is demonstrated as Aliamphritea ceti, hence, Aliamphritea hakodatensis could not described as gen. nov. sp. nov. but sp. nov. only.

B. The title could be changed as “Aliamphritea gen. nov. by genome based reclassification of the genus Amphritea and reclassification of Amphritea spongicola and Amphritea ceti as Aliamphritea spongicola comb. nov. and Aliamphritea ceti comb. nov. and Aliamphritea hakodatensis sp. nov.” or “Aliamphritea ceti gen. nov. comb. nov. and Aliamphritea spongicola comb. nov. via genome based reclassification of the genus Amphritea and Aliamphritea hakodatensis sp. nov.”

C. This is same for conclusion part.

2. About the description part

A. Description of the genus looks not fully adequate. In case of temperature, described as “mesotrophic” seems better and pH and salt response, basic components of the fatty acids or polar lipids, and respiratory quinone types could be included but DNA hydrolysis looks not. Additionally, parallel description of the isolation sources could be summarized as “marine animals” or describing as “marine animal related sources such as~” seems better.

B. Does there no changes in the description of two combined species compared to former description? Please check carefully and change.

3. About the genomic features : The authors described about PHB synthesis related genes in the core genes in two genera but nothing about function of core genes in each genus. Difference of genomic features could be a reason for dividing and important in the recognition of ecological role of two genera. Please including extensive analysis on genomic features of the two genera.

4. Others

A. Figure title seems not enough for fully understanding. Additional description or legend seems required.

i. Fig. 1 : Please include compared base no. and including descriptions on size marker and bootstrap values in the figure title.

ii. Fig. 2 : Actually it’s not very important and it could be combined with Fig. 3. Move to supplementary materials or remove but including values in the Fig. 3. And ANI values against type species could be included in the Table 1.

iii. Fig. 3 : As mentioned above, ANI values could be included in the half of the table format.

iv. Fig. 5 : Require explanation on color level.

v. Fig. 6 : Considering the (C) and (D), (E) could be looks like as (A) or (B). More explanation is required.

B. About Tables

i. Table 1 could be moved to supplementary materials.

ii. Table 2 : Please include other species also and explain “I” is what meaning?

iii. Additionally, genus level comparison is required at least genera included in the Fig. 4 for genus suggestion.

C. Minors

i. Line 26: among -> above

ii. Line 28 : Who’s AAI values? It’s not clearly described.

iii. Lines 89-90: What kind of method was used for distance calculation? It should be included with options. In this term, Lines 156-158 should be moved to “materials and method” part.

iv. Line 156: What is the K2+G+I model? Please described fully.

v. Line 185: Please provided original article as reference.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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2 Apr 2022

Dear Professor Vaz-Moreira, academic editor, and reviewers,

We appreciate editor and reviewers for constructive suggestions. We improved the manuscript PONE-D-21-39007 according to the reviewers’ comments. Responses for specific comments are described as follows. All changes were highlighted in yellow. Also, we noticed that tables were in graphic object form, so we changed them into editable objects.

Please see point-by-point response letter file uploaded in the last.

Submitted filename: Response_letter_PONE-D-21-39007.docx

Click here for additional data file.

26 Apr 2022

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

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If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Vyacheslav Yurchenko, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

The work of Yamano et al. was reviewed by 4 independent reviewers. All of them have indicated further modifications to the manuscript, which needs to be introduced in order to clarify several issues. I request the authors to address them all. The revised manuscript will be peer-reviewed once again. Importantly, please do not forget to upload supplementary materials this time.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

Reviewer #3: (No Response)

Reviewer #4: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Partly

Reviewer #4: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

Reviewer #3: N/A

Reviewer #4: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Major comment

The idea of "in silico chemotxonomy" is necessary as a new chemotxonomy, and the chemotxonomic information in this manuscript is described. However, it is necessary to discuss whether it can be used in a wide range of bacterial species.

I can't confirm because the supplementaly figure and table were not found.

Minor comment

P15-P24. There are many misspellings, so please reconfirm.

Table 1-3. Is it possible to summarize the order of species with Aliamphritea and Amphritea? Table 1-3 wants the order of the genera to be the same

Figures 6, 7, 8 and 9 should be moved to supplimentaly

Reviewer #2: The authors revised many but not enough for solving matters raised in previous review process. Details are as follows.

1. About the suggestion

A. As recommended, title was changed but same matters are remained in the abstract and main body of the MS.

i. Lines 34-41 also should be changed in accordance with the title. It can be suggested as follows; Depending on genome-based approaches, Aliamphritea gen. nov. was proposed with reclassification of the genus Amphritea and Aliamphritea ceti comb. nov. RA1T (=KCTC 42154T =NBRC 110551T), Aliamphritea spongicola comb. nov MEBiC05461T (=KCCM 42943T =JCM 16668T), and Aliamphritea hakodatensis sp. nov. PT3T(= JCM 34607T=KCTC 82591T). were suggested.

ii. Lines 58-63 : Please include the contents about dividing genus Amphritea into two genera.

iii. Conclusion part also should be changed.

B. Description part

i. Line 445 : remove “comb. nov.”

ii. Line 447 : remove “gen. nov.”

iii. Lines 450-451 and 457-458 : Genome of these species were newly reported, hence, including genome accession number is required.

iv. Line 482 : Provided number was not accession, please correcting it.

2. About the analysis of genome repertories

A. This MS pursuing reclassification of the genus Amphritea into two genera, hence, we will expecting the difference of genome repertories between two genera.

B. In this sense, lines 202-237 were good. And lines 243-250 also enough but Fig. 5 could be moved to supplementary materials.

C. However, lines 256-385 seems not important part of the analysis of genome repertories, these informs could be obtained by chemical analysis but ecological or physiological importance were unclear.

D. Hence, shorten the contents in lines 256-385 and strengthen the contents like in lines 202-237 (difference between two genera) seems better for readers.

3. About phenotypic comparison

A. Authors provided species by species comparison table, however, comparison at genus level is more important. Hence, Table S7 should be included in main body and Table 3 could be reduced within the range of genus Aliamphritea.

4. Others

A. Figures could be moved to supplementary or modified

i. Figs 5-6 : Could be moved to supplementary.

ii. Figs 7-9 : Moved to supplementary or simplified by bar plot type.

iii. Fig. 10 : Not very important and could be moved to supplementary.

B. About Tables

i. Table 1 : If not including that of strain PT3T it also moved to supplementary materials.

ii. Table 3 : Only members in Aliamphritea is enough.

C. Minors

i. Line 47: genera -> genus

ii. Lines 47-50 : Amphritea could be abbreviated as A. (eg A. japonica).

iii. Lines 72, 97, etc. : missing ‘,’.

iv. Lines 85 etc. : Check all PT3T in the MS, many times missing T and many times has interval between 3 and T.

v. Lines 179-182: “~ against above species~” seems enough.

Reviewer #3: Yamano et al. presented a manuscript regarding taxonomic revision of bacterial genus Amphritea. Establishing of the new genus Aliamphritea is justified by genomic analyses, which authors supplemented by chemotaxonomic analyses. Although this was a revised version, I still see a space for significant improvement, and several comments must be clarified before publication.

Major points:

I understand that the manuscript title was suggested by a reviewer, but I find it very long with unnecessary details. I suggest shortening it, e.g., “Taxonomic revision of the genus Amphritea supported by genomic and chemotaxonomic analyses”.

My major problem is lack of discussion. Although the chapter is named “Results and Discussion”, a large part of it is merely descriptive with very few biological inferences drawn from the results.

I find the usage of strain names an overkill. Surely, strain names should be clearly stated in methods, but to use them every time the species are mentioned is a bit excessive. Also, genus names should be abbreviated when used repeatedly.

Authors identified 1,660 gene clusters comprising 13,930 genes named “core”. They describe functions of very few of them on L208-211. Can authors elaborate on the rest of them?

Is difference of 10% in lipid content a significant change (Table 1)? Can those numbers be directly compared if they come from different studies? Did those studies use the same methodology? It is beyond my power to check methods in all mentioned references.

L146: Usage of chromosome 1 is not a “complete genomic sequence” as stated in the first part of the sentence. Please correct. Why did authors use only 1 chromosome of A. japonica?

Call out for Fig. 3 appears only in the legend of Fig. 2, but nowhere else in the manuscript. Comment on this figure in the main text. Moreover, I think Fig. 2 can be moved to supplement.

In Fig. 3 genes specific for a clade are called “Aliamphritea” and “Amphritea clade specific”, but in the text it is “Ceti” and “Atlantica clade specific”. Ceti and Atlantica also appears in Fig. S3. Please unify.

I would appreciate a composite figure of all synthetic pathways, i.e., adding Fig. S2 and S6 as panels to Fig. 6, and using the color codes for presence of genes in the two lineages (as in Fig. S2 now). If this was done, Table 2 could be moved to supplement.

I also do not see a point in showing genes for fatty acid, phospholipids and ubiquinone syntheses split into separate Fig. 7-9. Please combine them (using different colors for genes of different pathway). Right now, it seems that some genes from different pathway are overlapping (which may not be wrong).

In all tables, use species names instead of numbers and arrange species in a logical order (Aliamphritea species together, and Amphritea together).

L481-482: Accession numbers are not accessible. Also add corresponding accessions to supplementary tables even if they come from this study.

I think tables S3 and S4 can be merged into one listing which species were used for which analyses. Why not all species used for MLSA were used also for AAI calculations?

Fig. S1 in its current form is rather confusing. I have no idea how the numbers correspond to the species names. Can this be presented as a table?

Fig. S2 and S4 are not mentioned in the manuscript text. Please add call outs at appropriate places.

Although I consider typos and different formatting a minor thing, because of their extensive appearance throughout the whole text, I have decided to raise it as a major point. To name a few:

- L45: Oceanosprillales

- L97: missing comma

- L120 and L125: Ocenospirillaceae

- L176: missing genus names for A. opalenscence

- L224-225 and 419-421: incomplete sentences

- L273 and Table 1: Inconsistent usage of e.g. C16:0 as subscript with other parts of the text.

- Fig. 6: cis-3-decanoyl-ACP instead of cis-3-decenoyl-ACP

- L286-287: The last sentence of the paragraph is rather confusing. Rephase: “No genes responsible for the synthesis of ornithine-containing lipids were found in PT3 or any other Amphritea spp.”

Please read the whole manuscript carefully and fix typos, inconsistences, formatting, etc.

Minor points:

Abbreviations ANI, DDH, and AAI are not explained in the abstract. Please add their explanation.

Methods lack primer sequences that were used for PCR and/or sequencing.

Results and Discussion would hugely benefit from splitting the text to subchapters and adding appropriate titles.

L109-110: Include accession numbers for retrieved genome from NCBI.

L148-149: I believe that authors searched for enzymes of fatty acid, polar lipid, and isoprenoid quinone synthesis. The title should be corrected.

L152: What does “were mined from the genome sequences” mean? Proper description of tools and their parameters are necessary. Was BLAST or HMMER used? What version of the program? What was the Evalue cutoff threshold?

L185: If I correctly understand Fig. 2, the 93.7% corresponds to comparison of PT3 and A. spongicola, while 86.9% to PT3 and A. ceti. It is switched in the text.

L231: Similarly to one of my previous comments, genes do not encode putrescine. Please rephrase.

L295: Similarly, why is Phyre2 not mentioned in methods?

L267 and 311: Did authors mean linear FA by “straight-chained”?

L302: “previous chemotaxonomic properties” – Did author mean their results or previous studies? In any case, reference should be added.

L315: What is meant by “homogenous to”? Is it meant that genes of the core set in PT3 are homologous to other mentioned species?

Improve visualization of Fig. 5, right now it appears fuzzy.

What is “OF test” in Table 3? Also, “N” and “O” from the same row are not explained.

Reviewer #4: The collective Ryota Yamano et al. uses different approaches to establish the new genus Aliamphritea. The genomic data presented by authors are conclusive and original; however, before accepting the article, I suggest making a few changes.

Comments:

1. The chemotaxonomic characteristics summarized in Table 1 represents published data and is therefore not original, so I propose to move it to supplementary data.

2. Table 1: marking the species with names instead of numbers would make the table more readable.

3. I suggest improving the discussion, which is not visible between the results.

4. Unify the naming of the new species. It is recognized as PT3 in Fig 4., and Fig.7; Aliamphritea hakodatensis in Fig.3, and both names are used in Fig. 1 and Fig. 2.

5. Line 267: straight-chain change to linear-chain.

6. Fig S2. Predicted polyamine metabolism pathways in Aliamphritea and Amphritea: change to “Predicted polyamine metabolism pathways in Aliamphritea and Amphritea species.”

7. Fig S6. Predicted Q-8 synthetic pathway in Amphritea and Aliamphritea: change to “Predicted Q-8 synthetic pathway in Amphritea and Aliamphritea species. “

8. Accession numbers of genes originated from this study should be added into all tables instead of “in this study”.

9. Table S2. Genomic properties of Aliamphritea and Amphritea species: see previous point. Change Alaimphritea to Aliamphritea in the first two rows.

10. Table S3. List of genomes used for AAI calculation: see the point 8.

11. Table S7. Phenotypic characteristics of Aliamphritea and genus level comparison within the family Oceanospirillaceae: Oeanospirillum change to Oceanospirillum

Before publishing, I recommend another round of thorough review to minimize typographical errors.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

9 May 2022

Dear Professor Yurchenko, the academic editor, and reviewers,

We appreciate editor and reviewers for constructive suggestions. We improved the manuscript according to the reviewers’ comments. Responses for specific comments are described as follows. All changes were highlighted in yellow.

Please see details in the point-by-point response letter uploaded separately.

best

Tomoo Sawabe

Submitted filename: Response_letter_May7_Yamano_D-21-39007R2.docx

Click here for additional data file.

30 May 2022

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Vyacheslav Yurchenko, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

The manuscript was reviewed by 2 independent referees and they both recommended further improvements. I side with all their critique and request another round of major revision. Restructuring and straightening the manuscript also seem like good ideas to me. Please note the manuscript will be peer-reviewed again.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Partly

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: N/A

Reviewer #3: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: No

Reviewer #3: No

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: The authors revised many and now could comments more about taxonomic points. In addition, English should be improved. Contents on genomic repertories also described clearly by separating common, original, and ceti group. Details are as follows.

1. Suggestion on MS structure and order of the contents in the “Results and Discussion” part

A. Divide the results into two parts, one for basic reclassification and another for further works on genomic contents.

i. In the MS, Aliamphritea mentioned before suggestion of the novel genus, it makes confusion. Hence, suggestion of the genus Aliamphritea based on phylogenetic and OGRIs results conducted first. Then, describe genome based analysis results.

ii. After suggestion of the genus name, ‘Aliamphritea Core” or “Amphritea Core” makes no problems. If not, presented as “Aliamphritea core -> ceti group core & Amphritea core -> atlantica group core” is adequate.

iii. Additionally, “Core” including Amphritea and Aliamphritea should be changed to “Common”.

B. Range of comparison in the “Phenotypic Characterization”

i. If suggesting novel genus at the first half of the Results part, phenotypic comparison between species could be reduced to species in the novel genus. As it say, Table 2 composed with first 3 species is enough. Table of this time could be given as supplementary. Of course, discussion part (lines 451-466) should be amended.

ii. Additional requirements for suggestion of novel genus: For suggestion of novel genus, genus level comparison should be given. In this case, including all genera in the family is normal process. However, Oceanospirillaceae is too big, hence, at least, compare with phylogenetically close genera such as Amphritea, Corallomonas, Neptuniibacter, Neptunomonas, Pontibaculum, Profundimonas, and Oceanispirillum is recommended. In this case, table includes basic features described in the genus is enough.

C. Possible combining or addition

i. Table S1 and S2 could be combined with all information. These two are actually overlap but inform given each is not enough.

ii. Fig. 5 : For demonstrate author’s suggestion, more plots including intra groups (atlantica vs. opalescens, japonica vs. balenae) should be given. Additionally, if evolution by inversion is proved, Fig. S3 should be moved to main text and provided by combined with Fig. 5.

iii. Fig. 6 : Genes present or not depend on species or group should be given. At present general pathway only.

2. Others

A. Require amendments

i. 16S rRNA gene based phylogeny (Fig. 1): Trimming inform and number of compared base should be given.

ii. Line 192: Need some discussion about this. Why ceti group has bigger genome?

iii. Title of Fig. 3: Of course, gene sequences from genome, it’s not genome tree. Hence, line 218 is not adequate.

B. Minors

i. Lines 26 and 120: (in silico DDH) -> (isDDH)

ii. Lines 33-34: (PT3T, A. ceti RA1T and A. spongicola MEBiC05461T) is enough

iii. Lines 37-39: change like as this “~ sp. nov. (type strain RA1T =KCTC ~)

iv. Line 106: manufactureR

v. Line 114: this is not actual accession number but “assembly accession” number. Pls change.

vi. Line 120: Average “NUCLEOTIDE” Identity

vii. Line 357: Des4 is not common, hence, need to rephrase.

viii. Line 482: 42943T), RESPECTIVELY.

ix. Line 482: novel genus -> genus Aliamphritea

x. Line 483: remove “gen. nov.”

xi. Line 498: Remove strain name, species name is enough in here.

xii. Lines 501 & 511: remove “,” after “et al.”

xiii. Line 521: insert “and: after “cream”

Reviewer #3: The authors addressed majority of the reviewers’ points, but unfortunately not all of them:

- I suggested to remove strain names from the manuscript (except for Methods). Authors indeed did so but starting by subchapter “Pan and core genome analysis”, previous subchapters still contain strain names.

- Me and reviewer #2 suggested to abbreviate species names (e.g., Amphritea to A. and Aliamphritea to Al.), but authors did not comply, because it could be confused with sea cucumber Apostichopus japonicus. I still think that abbreviating bacterial names, while keeping sea cucumber in full, would increase clarity of the text.

- One of the sentences that was incomplete remained incomplete (L464-466 now).

- Species names were not added to the tables in main text. Tables would be much clearer with them instead of numbers.

Altogether, further improvements are needed before manuscript can be considered for publication. I also raise new points that should be addressed.

New comments:

- Unify writing of thousands (with or without comma) throughout the text.

- L97: rephrase -> “were calculated using the K2 model in MEGAX as well.”

- L130: Table S2 is mentioned before Table S1 (L192), please correct.

- L162-164: rephrase -> “3D-structure of FA desaturase encoding genes from some

of the strains was predicted by Phyre2 [27].”

- L176: typo “linages” -> “lineages”

- L180: correct “which IS frequently used”

- L198: What is “Formula2”?

- L198-199: Where are DDH values shown? Is there some figure or table? Refer to it please.

- L207: According to Fig. 2, AAI value for PT3 and A. atlantica is 64.6%, so this number should be shown in the range.

- L228-229: “3-hydrokyalkanoate” -> “3-hydroXyalkanoate”

- L264: Can authors unify the use of “-aminobutyrate” in text and “4-aminobutanoate” in Fig. S2?

- L276: Call out of Fig. 4 is not appropriate here, there are no particular genes shown in this figure.

- L320 and L323: I believe it should be “comprising” or “making up” instead of “consisting”.

- L359-360: “found from” -> “found in”

- L381: Please check call outs for figure and table here, it does not look correct.

- L404-405: “which is responsible for octaprenyl-diphosphate synthesis” repeats information from two lines above.

- L406: missing comma after ubiJ

- L406: These essential genes are not present in the scheme in Fig. S9. Can authors describe their function at least in the text?

- L411: Fig. S8 is mentioned after Figs. S9 (L401) and S10 (L408), please correct.

- L423: belonged -> belonging

- L438: “PT3Twas” – missing space

- L440, 443, Table 2: Unify “Tween80”, “tween 80”, “Tween 80”.

- L447: I believe that authors wanted to say “diagnostic features […] differing from other genera were found”. If not, rewrite to make it clear.

- L484-485: Move accession numbers to Methods, there is no need for them in Conclusions.

- Fig. 3: Remove underscores from and add italics to species names.

- Fig. S3: Text of Amphritea species is fuzzy and underlined. Please correct.

- Fig. S4, S7, and S8: Can authors improve the quality of these figures?

- Table 2: *1 and *2 should be presented as superscripts.

- Table S3: *1 and *2 are not present in the table, but are explained below the table. Please, correct.

- Table S4: Typos in species names A. balenae and A. japonica.

- Table S5: Typo in Al. ceti.

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Reviewer #3: No

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14 Jun 2022

Dear editor and reviewers,

We appreciate editor and reviewers for constructive suggestions. We improved the manuscript according to the reviewers’ comments. Responses for specific comments are described in a separate file. All changes were highlighted in yellow.

Submitted filename: Response_letter_14June22.docx

Click here for additional data file.

27 Jun 2022

Taxonomic revision of the genus Amphritea supported by genomic and in silico chemotaxonomic analyses, and the proposal of Aliamphritea as a gen. nov.

PONE-D-21-39007R3

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Reviewer #4: All comments have been addressed

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Reviewer #4: Yes

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Reviewer #4: I Don't Know

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Reviewer #4: (No Response)

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19 Jul 2022

PONE-D-21-39007R3

Taxonomic revision of the genus Amphritea supported by genomic and in silico chemotaxonomic analyses, and the proposal of Aliamphritea gen. nov.

Dear Dr. Sawabe:

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