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.
Citation: Yamano R, Yu J, Jiang C, Harjuno Condro Haditomo A, Mino S, Sakai Y, et al. (2022) Taxonomic revision of the genus Amphritea supported by genomic and in silico chemotaxonomic analyses, and the proposal of Aliamphritea gen. nov. PLoS ONE 17(8): e0271174. https://doi.org/10.1371/journal.pone.0271174
Editor: Vyacheslav Yurchenko, Ostravska univerzita, CZECH REPUBLIC
Received: December 9, 2021; Accepted: June 23, 2022; Published: August 10, 2022
Copyright: © 2022 Yamano et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The GenBank accession number for the 16S rRNA gene sequence of the type strain is OL455018. The whole genome sequence of the strain has been deposited to DDBJ/ENA/GenBank under the accession number AP025281-AP025284, and PRJDB12633.
Funding: This study was supported by Kaken 19K22262. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
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 . Subsequently, six species have been proposed in this genus: Amphritea japonica, and Amphritea balenae from the sediment adjacent to sperm whale carcasses , Amphritea ceti from Beluga whale feces , Amphritea spongicola from a marine sponge , Amphritea opalescens from marine sediments  and Amphritea pacifica from a mariculture fishpond . 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 , which has been suggested as contributing to growth gaps in the sea cucumber Apostichopus japonicus . 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 . 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)  and NCBI databases. Sequences were aligned using Silva Incremental Aligner v1.2.11 . 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 . 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) . 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  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 , 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 .
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 . Concatenation of sequences and phylogenetic reconstruction were performed using SplitsTree 4.16.2 .
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  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.
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 .
Results and discussion
Molecular phylogenetic analysis based on 16S rRNA gene nucleotide sequences
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 , 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].
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 . 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% . 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% . The AAI values indicated that PT3T, A. spongicola and A. ceti could affiliate to a novel genus.
Reference genomes were downloaded from NCBI database (S2 Table).
Multilocus sequence analysis (MLSA)
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.
A list of strains and their assembly accession is provided in S2 Table.
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.
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.
Pan and core genome analysis
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.
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 . 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 . 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 . In particular, GABA is a neurotransmitter widely distributed in animals including sea cucumber , which indicates that Aliamphritea species might influence the nervous system of their host during the developmental stage . 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 . 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 , 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 . 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 . 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 .
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).
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  (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 . 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 . 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.
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.
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 . 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) . 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 .
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 .
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 , 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) , 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) . 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 . 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 . The in silico chemotaxonomy approach is one way to achieve this, and this methodology has been used exclusively in Corynebacterium and Turicella . 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.
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 .
The description is the same as that published for Amphritea ceti by Kim et al. (2014) . 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 .
The description is the same as that published for Amphritea spongicola by Jang et al. (2015) . 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).
S1 Fig. Heat map representation of ANI values of Aliamphritea and Amphritea species.
S2 Fig. An electron micrograph of negatively stained Aliamphritea hakodatensis PT3T cell.
The bar represents 1 μm.
S3 Fig. Predicted polyamine metabolism pathways in Aliamphritea and Amphritea species.
S4 Fig. Evolutionary history of Aliamphritea and Amphritea genome arrangement.
S5 Fig. Amino acid sequence alignment of Des1-4.
S6 Fig. 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.
S7 Fig. Genomic structure of Amphritea and Aliamphritea fab and associated genes.
S8 Fig. 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.
S9 Fig. Predicted Q-8 synthetic pathway in Amphritea and Aliamphritea species.
S10 Fig. Genomic structure of ubi and associated genes.
S11 Fig. 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.
S1 Table. Genome properties of Aliamphritea and Amphritea species.
S2 Table. List of other Oceanospirillaceae genomes used for genome taxonomy of Aliamphritea and Amphritea.
+: used, -: not used.
S3 Table. 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.
S4 Table. Results of 3D-structure prediction of Des1-4 by Phyre2.
S5 Table. PG, PE and DPG associated genes composition of each strain.
+: genes presence; -: absence.
We gratefully thank for Professor (Emeritus) Aharon Oren, The Hebrew University of Jerusalem, for his advice on bacterial names.
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