Genome taxonomy of the genus Neptuniibacter and proposal of Neptuniibacter victor sp. nov. isolated from sea cucumber larvae

Rika Kudo; Ryota Yamano; Juanwen Yu; Shotaro Koike; Alfabetian Harjuno Condro Haditomo; Mayanne A M de Freitas; Jiro Tsuchiya; Sayaka Mino; Fabiano Thompson; Jesús L Romalde; Hisae Kasai; Yuichi Sakai; Tomoo Sawabe

doi:10.1371/journal.pone.0290060

. 2023 Aug 15;18(8):e0290060. doi: 10.1371/journal.pone.0290060

Genome taxonomy of the genus Neptuniibacter and proposal of Neptuniibacter victor sp. nov. isolated from sea cucumber larvae

Rika Kudo ¹, Ryota Yamano ¹, Juanwen Yu ¹, Shotaro Koike ¹, Alfabetian Harjuno Condro Haditomo ^1,², Mayanne A M de Freitas ³, Jiro Tsuchiya ¹, Sayaka Mino ¹, Fabiano Thompson ³, Jesús L Romalde ⁴, Hisae Kasai ¹, Yuichi Sakai ⁵, Tomoo Sawabe ^1,^*

Editor: Karel Sedlar⁶

¹Laboratory of Microbiology, Faculty of Fisheries Sciences, Hokkaido University, Hakodate, Japan

²Aquaculture Department, Faculty of Fisheries and Marine Sciences, Universitas Diponegoro, Semarang, Indonesia

³Laboratory of Microbiology, Biology Institute, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil

⁴Departamento de Microbiología y Parasitología, CRETUS & CIBUS-Facultad de Biología, Universidade de Santiago de Compostela, Santiago, Spain

⁵Hakodate Fisheries Research, Hokkaido Research Organization, Local Independent Administrative Agency, Hakodate, Japan

⁶Brno University of Technology: Vysoke uceni technicke v Brne, CZECH REPUBLIC

Competing Interests: The authors have declared that no competing interests exist.

^✉

* E-mail: sawabe@fish.hokudai.ac.jp

Roles

Rika Kudo: Data curation, Formal analysis, Methodology, Writing – original draft, Writing – review & editing

Ryota Yamano: Data curation, Formal analysis, Methodology, Writing – original draft, Writing – review & editing

Juanwen Yu: Conceptualization, Data curation, Formal analysis, Project administration, Writing – original draft, Writing – review & editing

Shotaro Koike: Data curation, Methodology, Writing – original draft, Writing – review & editing

Alfabetian Harjuno Condro Haditomo: Formal analysis, Methodology, Writing – review & editing

Mayanne A M de Freitas: Formal analysis, Methodology, Writing – original draft, Writing – review & editing

Jiro Tsuchiya: Data curation, Methodology, Writing – review & editing

Sayaka Mino: Conceptualization, Data curation, Funding acquisition, Methodology, Supervision, Writing – review & editing

Fabiano Thompson: Writing – review & editing

Jesús L Romalde: Resources, Writing – review & editing

Hisae Kasai: Writing – review & editing

Yuichi Sakai: Conceptualization, Investigation, Supervision, Writing – review & editing

Tomoo Sawabe: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing

Karel Sedlar: Editor

PMCID: PMC10426996 PMID: 37582072

Abstract

A Gram-staining-negative, oxidase-positive, strictly aerobic rod-shaped bacterium, designated strain PT1^T, was isolated from the laboratory-reared larvae of the sea cucumber Apostichopus japonicus. A phylogenetic analysis based on the 16S rRNA gene nucleotide sequences revealed that PT1^T was closely related to Neptuniibacter marinus ATR 1.1^T (= CECT 8938^T = DSM 100783^T) and Neptuniibacter caesariensis MED92^T (= CECT 7075^T = CCUG 52065^T) showing 98.2% and 98.1% sequence similarity, respectively. However, the average nucleotide identity (ANI) and in silico DNA-DNA hybridization (DDH) values among these three strains were 72.0%-74.8% and 18.3%-19.5% among related Neptuniibacter species, which were below 95% and 70%, respectively, confirming the novel status of PT1^T. The average amino acid identity (AAI) values of PT1^T showing 74–77% among those strains indicated PT1^T is a new species in the genus Neptuniibacter. Based on the genome-based taxonomic approach, Neptuniibacter victor sp. nov. is proposed for PT1^T. The type strain is PT1^T (JCM 35563^T = LMG 32868^T).

Introduction

The genus Neptuniibacter, belonging to the family Oceanospirillaceae, was first proposed by Arahal et al. (2007) with Neptuniibacter caesariensis, which was isolated from surface water from the Eastern Mediterranean [1]. Currently, four species including N. caesariensis have been described in this genus: Neptuniibacter halophilus, isolated from a salt field in southern Taiwan [2], Neptuniibacter marinus and Neptuniibacter pectenicola, both from a scallop hatchery in Norway [3]. Members of the genus Neptuniibacter have been isolated from seawater and/or salt water, and the genus is characterized as being Gram-staining negative, motile, oxidase-positive, strictly aerobic rods requiring NaCl and/or sea salts [1–3].

In the process of collecting reference genomes to understand the structure, function, and dynamics of the sea cucumber microbiota [4–6], the strain PT1^T was isolated from the larvae of Apostichopus japonicus [4]. The strain PT1^T possesses similar sequences to those of the key Amplicon Sequence Variant (ASV0001-0004), which significantly increased its abundance during the pentactula and juvenile stages found in meta16S analyses of the early developmental stages in sea cucumber [4]. The strain PT1^T could have previously unknown interactions with the host sea cucumber. Moreover, PT1^T is the only isolate identified to the genus Neptuniibacter among 237 isolates obtained from the early life stage of A. japonicus [4]. Due to the absence of comprehensive studies on the genomic characterization of the genus Neptuniibacter and fewer animal-associated Neptuniibacter isolates, genome fundamentals can contribute not only to host-microbes interaction studies but also to genome taxonomy. Here we report the genome characterization of the newly described Neptuniibacter sp. strain PT1^T, and propose it as Neptuniibacter victor sp. nov. based on the genome taxonomy.

Materials and methods

Bacterial strains and phenotyping

The strain PT1^T was isolated from the pentactula larvae of A. japonicus reared in a laboratory aquarium in July 2019 [4]. In brief, larvae were collected using 40 μm nylon mesh (FALCON Cell Strainer, Durham, USA), washed once with sterilized seawater, and then manually homogenized in 1 mL filter-sterilized natural seawater for 60 seconds. Ten-fold serial dilutions of the homogenate were cultured on 1/5 strength ZoBell 2216E agar plates [4]. Bacterial colonies were purified using the same agar plates. After purification, the isolate was stored at -80°C suspended in glycerol supplemented 1/5 strength ZoBell 2216E broth. N. halophilus LMG 25378^T, N. caesariensis CECT 7075^T, N. marinus CECT 8938^T, N. pectenicola CECT 8936^T were used for genomic and phenotypic comparisons against the strain PT1^T. All strains were cultured on Marine agar 2216 (BD, Franklin Lakes, New Jersey, USA). The phenotypic characteristics were determined according to previously described methods and Traitar approach [5–10]. Motility was observed under a microscope using cells suspended in droplets of sterilized 75% artificial seawater [5, 6]. API20 NE (bioMérieux, Craponne, France) was also used to examine phenotypes according to Chen et al. [2].

Due to difficulties in the growth of PT1^T in manually prepared marine Oxidative/Fermentative (OF), a salt requirement test, and synthetic basal marine media for evaluating carbon assimilations, we also used the in silico phenotyping tool, Traitar ver. 1.1.2 (Microbial Trait Analyzer) [11], instead of the experimental approach to compare predicted phenotypes based on genome sequences among Neptuniibacter species. This software is capable of predicting 67 phenotypic traits using Prodigal for gene prediction and Pfam family for annotation [11]. The software uses two prediction models, the phypat model (which predicts the presence/absence of proteins found in the phenotype of 234 bacterial species) and a combination of phypat+PGL models (which uses the information of phypat combined with the information of the acquisition or loss of protein families and phenotypes through evolution), to determine the phenotypic characteristics [11].

Whole genome sequencing

Genomic DNAs of PT1^T and N. halophilus were extracted from 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 a flow cell (FLO-MIN 106), and a 48-hour sequencing run with MinKNOW ver. 3.6.0 software was performed. Basecalling was carried out using Guppy ver. 4.4.1 (Oxford Nanopore Technologies). For Illumina sequencing, a 300 bp paired-end library was prepared using the NEBNext Ultra II FS DNA Library Prep Kit. The ONT and Illumina reads were assembled using Unicycler ver. 0.4.8 [12]. After checking quality value (QV) of these Illumina reads over 27 with no adaptor and barcode sequences assessed using FastQC program ver. 0.11.9 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/), these reads were used for genome assembly without preprocessing. Genome sequences of N. caesariensis, N. marinus, and N. pectenicola were retrieved from the NCBI database; the accession numbers are GCF_000153345.1, GCF_001597725.1, and GCF_001597735.1 [1, 3]. The whole genome sequences were annotated with DDBJ Fast Annotation and Submission Tool (DFAST) [13]. The complete genome sequences of PT1^T and N. halophilus LMG 25378^T acquired in this study were deposited in GenBank/EMBL/DDBJ under accession number AP025763 and AP027292-AP027293, respectively.

Molecular phylogenetic analysis based on 16S rRNA gene nucleotide sequences

The full-length 16S rRNA gene sequence of strain PT1^T was obtained from the complete genome sequence. The 16S rRNA gene nucleotide sequences of the type strains of the genus Neptuniibacter and other Oceanospirillaceae species were retrieved from NCBI databases. Sequences were aligned using MEGA X ver. 11.0.11 [14]. A phylogenetic model test and reconstruction of maximum likelihood (ML) tree were performed using the MEGA X ver. 11.0.11 [14, 15]. 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 MEGA X software [14].

Overall genome relatedness indices (OGRIs)

Overall genome relatedness indices (OGRIs) were calculated to determine the novelty of PT1^T using same approach by Yamano et al. [5, 6]. Average nucleotide identities (ANIs) were calculated using the Orthologous Average Nucleotide Identity Tool (OrthoANI) software ver. 0.93.1 [16] using genomes of the PT1^T and previously described Neptuniibacter type strains. In silico DNA-DNA hybridization (DDH) values were calculated using Genome-to-Genome Distance Calculator (GGDC) ver. 2.1 with formula 2, being the most robust against incomplete genomes [17]. Average amino acid identities (AAIs) were calculated and compared between PT1^T and Neptuniibacter species (Table 1) using an enveomics toolbox [18].

Table 1. List of genomes used in this study.

Species	Strain	RefSeq/GenBank accession	Size (bp)	No. Of contigs	G+C content	MLSA	AAI
*Neptuniibacter victor*	PT1 ^T	AP025763 (in this study)	3,952,146	1	44.2%	+	+
*Neptuniibacter halophilus*	LMG 25378 ^T	AP027292-AP027293 (in this study)	4,113,600	2	53.3%	+	+
*Neptuniibacter caesariensis*	MED92 ^T	GCF_000153345.1	3,924,755	6	46.0%	+	+
*Neptuniibacter marinus*	ATR 1.1 ^T	GCF_001597735.1	3,454,191	117	42.8%	+	+
*Neptuniibacter pectenicola*	LFT 1.8 ^T	GCF_001597725.1	3,631,894	203	45.7%	+	+
*Aliamphritea ceti*	RA1 ^T	AP025282	5,213,210	1	47.1%	+	-
*Aliamphritea hakodatensis*	PT3 ^T	AP025281	5,208,344	1	52.2%	+	-
*Aliamphritea spongicola*	MEBiC05461 ^T	AP025283	4,964,135	167	51.5%	+	-
*Amphritea balenae*	JAMM1525 ^T	GCF_014646975.1	4,671,747	18	47.7%	+	-
*Amphritea pacifica*	ZJ14W ^T	GCF_016924145.1	4,702,884	282	51.2%	+	-
*Amphritea atlantica*	M41 ^T	AP025284	4,804,482	1	51.1%	+	-
*Amphritea japonica*	JAMM1866 ^T	AP025761-AP025762	3,880,036	2	47.6%	+	-
*Amphritea opalescens*	ANRC-JH13 ^T	GCF_003957515.1	4,124,452	46	48.5%	+	-
*Neptunomonas antarctica*	S3-22 ^T	GCF_900156635.1	4,569,005	25	45.7%	+	-
*Neptunomonas phycophila*	Scap09	GCF_013394205.1	3,976,465	1	45.5%	+	-
*Marinomonas arctica*	BSI20414	GCF_014623465.1	4,540,024	1	44.5%	+	-
*Marinomonas mediterranea*	MMB-1 ^T	GCF_000192865.1	4,684,316	1	44.1%	+	-
*Marinomonas posidonica*	IVIA-Po-181 ^T	GCF_000214215.1	3,899,940	1	44.3%	+	-
*Oceanospirillum beijerinckii*	NBRC 15445 ^T	GCF_000422425.1	5,325,321	99	47.8%	+	-
*Oceanospirillum maris*	ATCC 27509 ^T	GCF_000422865.1	3,709,807	44	45.9%	+	-
*Oceanospirillum multiglobuliferum*	NBRC 13614 ^T	GCF_900167095.1	3,512,709	46	45.4%	+	-
*Marinobacterium aestuarii*	ST58-10 ^T	GCF_001651805.1	5,191,608	1	58.8%	+	-
*Marinobacterium litorale*	DSM 23545 ^T	GCF_000428985.1	4,380,400	64	56.4%	+	-
*Marinobacterium mangrovicola*	DSM 27697 ^T	GCF_004339595.1	4,979,947	15	57.1%	+	-
*Nitrincola lacisaponensis*	4CA ^T	GCF_000691225.1	3,412,133	43	52.1%	+	-
*Nitrincola tapanii*	MEB 193 ^T	GCF_008368715.1	2,793,747	19	50.8%	+	-
*Nitrincola tibetensis*	xg18 ^T	GCF_003284585.1	4,001,852	54	46.1%	+	-

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+: used, -: not used.

Multilocus sequence analysis (MLSA)

MLSA including concatenation of sequences and phylogenetic network reconstruction were performed using SplitsTree ver. 4.16.2 with the almost same setting [5–10, 19]. The sequences of four protein-coding genes (mreB, recA, rpoA, and topA) were obtained from the genome sequences of PT1^T, N. halophilus LMG 25378^T, N. caesariensis CECT 7075^T, N. marinus CECT 8938^T, N. pectenicola CECT 8936^T and other related Oceanospirillaceae species (Table 1). These sequences were aligned using Clustal X ver. 2.1 [20]. Regions used for the network reconstruction in Fig 2 were 1–995, 1–1,041, 1–981, and 1–2,648 for mreB, recA, rpoA, and topA, as PT1^T nucleotide sequence positions (GenBank accession number AP025763), respectively.

Fig 2 — A list of strains and their assembly accession is provided in Table 1. Sequence regions used for reconstructing the network were 1–995 on *mreB*, 1–1,041 on *recA*, 1–981 on *rpoA*, and 1–2,648 on *topA*. The clade which PT1^T belonged was robust with supported by 100% bootstrap.

Pan and core genome analyses

A total of five genomes, including two newly obtained in this study (PT1^T and N. halophilus LMG 25378^T) and three retrieved from the NCBI database (N. caesariensis, N. marinus, N. pectenicola) were used for pan- and core-genome analyses using the program anvi’o ver. 7 [21] based on previous studies [5, 6, 10, 22], with minor modifications. Briefly, contig 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 a 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.

In silico chemical taxonomy

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 PT1^T and four previously described Neptuniibacter species using in silico MolecularCloning ver. 7 (In Silico Biology, Yokohama, Japan) based on the same approach of Yamano et al. [5, 6]. The structure and distribution of the genes were also compared using in silico MolecularCloning ver. 7. The 3D-structure of FA desaturase gene products found in Neptuniibacter strains was predicted using Phyre2 ver. 2 [23] with the same approach described previously [5, 6].

Results and discussion

Molecular phylogenetic analysis based on 16S rRNA gene nucleotide sequences

Phylogenetic analysis based on 16S rRNA gene nucleotide sequences showed that the strain PT1^T was affiliated to the members of the genus Neptuniibacter showing 96.7–98.2% sequence similarities, which are below the proposed threshold range for species boundary at 98.7% [24, 25]. The strain showed high sequence similarities of 98.2% with N. marinus. The tree also revealed that the PT1^T formed a robust monophyletic cluster with N. caesariensis, N. halophilus, N. marinus, and N. pectenicola within the genus Neptuniibacter (Fig 1).

The ML tree was reconstructed using the K2+G+I model. Numbers shown on branches are bootstrap values (%) based on 1,000 replicates (>70%). A total of 1,284 bp was compared (162–1,446 position in PT1^T, GenBank accession number AP025763). 16S rRNA gene nucleotide sequences loci r00010 and r00040 under GenBank accession number GCF_000974885.1 were used as an outgroup to generate this rooted tree. Bar, 0.05 substitutions per nucleotide position.

Genomic features and overall genome relatedness indices (OGRIs)

The ANI values of the PT1^T against N. halophilus, N. caesariensis, N. marinus, and N. pectenicola were 72.0%, 72.4%, 74.8%, and 74.7%, respectively (S1 Fig), which are below the species boundary threshold of 95% proposed in previous studies [26]. The DDH values of PT1^T against those species were 18.7%, 18.3%, 19.5%, and 19.1%, respectively, which were also below the species delineation threshold (70%). ANI and in silico DDH revealed PT1^T as a novel species.

The AAI values of PT1^T for against N. halophilus, N. caesariensis, N. marinus, and N. pectenicola were 75.8%, 74.8%, 77.8%, and 77.7%, respectively, which are below the 95% species boundary and above the 64–67% genus boundary, indicating that they are new species within the genus (S2 Fig) [5, 6, 8, 27].

Multilocus sequence analysis (MLSA)

MLSA network showed that PT1^T is likely to be monophyletic with previously described Neptuniibacter species but conspecifically separate from them. This result supports the proposal that PT1^T is a novel species in the genus Neptuniibacter (Fig 2).

Experimental phenotypic characterization and in silico phenotyping

Several experimental phenotypic characterization tests revealed that PT1^T shares several characteristics with Neptuniibacter spp. including being positive for oxidase and growth at 15°C and 30°C [1–3]. PT1^T could be differentiated from Neptuniibacter spp. by growth at 4°C, 10°C, and 37°C, catalase reaction, hydrolysis of starch and DNA, nitrate reduction, and utilization of glucose, and DL-malic acid (Table 2).

Table 2. Phenotypic characteristics of PT1^T and Neptuniibacter strains.

Characteristics	N. victor sp. nov. PT1^T	N. halophilus^T	N. caesariensis^T	N. marinus^T	N. pectenicola^T
Growth at
4°C	-	-	-	+	+
10°C	+	-	+	+	+
37°C	-	+	+	-	+
45°C	-	-	-	-	-
Catalase	-	-	+	-	+
Production of
amylase	-	-	-	+	-
DNase	w	w	-	nd	nd
Nitrate reduction	+	-	-	-	-
Utilization of
D-glucose	+	-	-	-	-
DL-malic acid	-	-	+	+	+

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Phenotypic data for Neptuniibacter are from this and previous studies [1–3]. All strains are positive in growth at 15–30°C, oxidase test. All strains are negative in growth at 45°C, indole production, utilization of L-arabinose, D-mannitol, N-acetyl-D-glucosamine, maltose, and hydrolysis of Tween 20 and 80, and gelatin.

+: present; -: lack; w: weak reaction; nd: not determined.

Unfortunately, PT1^T was unable to be grown in manually prepared media commonly used for OF, salt requirement, and carbon assimilation tests [5–8, 10], so we also use in silico phenotyping approach using Traitar software for predicting the phenotype. Before predicting PT1^T phenotype, the accuracy of Traitar phenotyping was evaluated to be 81.5% in average using phenotypic characterization from four described Neptuniibacter species (N. halophilus, N. caesariensis, N. marinus, and N. pectenicola) with 40–47 traits compared. The Traitar prediction showed that PT1^T was differentiated by nine traits among Neptuniibacter species: utilization of acetate (accuracy 50%), cellobiose (accuracy 100%), maltose (accuracy 75%) and mucate (not determined), hydrolysis of casein (accuracy 50%), production of alkaline phosphatase (accuracy 75%), indole (accuracy 75%), lysine decarboxylase (not determined), and growth at 42°C (accuracy 25%) (S3 Fig). However, no maltose utilization and indole production were observed using the API20NE.

Pan and core genomes and its ecogenomic interpretation

The pangenome of Neptuniibacter species consists of 6,605 gene clusters (17,748 genes) (Fig 3). Genes were classified into Core, present in all strains, and Unique in individual species. Core consisted of 1,973 gene clusters (10,215 genes). COG categories such as J (translation), K (transcription) and E (amino acid transport) were abundant in this bin. Unique in PT1^T consists of 596 gene clusters (612 genes). COG categories such as E (amino acid transport system) and I (lipid transport and metabolism) were included in this bin. Genes related to nitrogen metabolism were also detected in Unique in PT1^T.

Interestingly, PT1^T possessed a 45 kb gene island encoding a type VI secretion system (T6SS) and genes (narGHJI and norBC) responsible for performing several steps of denitrification, nitrate and nitric oxide reductions. These genes were grouped into the PT1-Unique genes by pangenomic analysis. Recently, T6SS has been identified as having a role in killing phenotypes of Vibrio fischeri to establish spatial separation in different crypts of a light organ in the host Euprymna scolopes squid [28, 29]. NarGHI is typically identified as a dissimilatory nitrate reductase with a role in nitrate respiration under not only anaerobic but also aerobic conditions [30, 31]. The presence of formate dehydrogenase genes suggests that formate could be served as an electron donor for nitrate respiration in PT1^T [30]. Nitrite is further metabolized by a nitrate reductase NirBD to ammonia in the PT1^T. The assimilatory nitrate reduction pathway using NasAC and NirA has also been predicted in this strain [32]. Detoxification of nitric oxide (NO), which is a multifunctional immune trigger involving antimicrobial activity, using a nitric oxide reductase, NorBC, has been known as a function in sustaining host-microbes interactions in a wide variety of animals and plants [33]. As PT1 possesses similar sequences to those of the key abundant ASVs associated with the pentactula larvae of A. japonicus, the T6SS with nitrate respiration and NO detoxification genotypes of the PT1^T could contribute to competitive dominance in the early life of the benthic host animal. Genes responsible for poly-β-hydroxybutyrate biosynthesis were found in the PT1^T genome [34], but the effects of the PT1^T on promoting growth in sea cucumber A. japonicus have yet to be observed (data not shown).

In silico chemical taxonomy

As fatty acids, polar lipids, and isoprenoid quinones are common subjects for chemical taxonomic analyses, in silico chemical taxonomy among the Neptuniibacter species including the newly described species of PT1^T based on genome sequences, as an alternative to the traditional chemical taxonomy, was performed (S1 Table).

Comparative genome analyses among described Neptuniibacter species referring reported cellular fatty acids of Neptuniibacter species, which are mainly linear, mono-unsaturated or saturated consisting of C16:0, C16:1 and C18:1, with a small amount of C10:0 3-OH (S1 Table) [1–3], reconstructed the basic type II fatty acid biosynthesis (FAS II) pathway driven by fabABFDGIVYZ, which is very similar to that of E. coli [35] (S4–S6 Figs). In particular, long-chain saturated fatty acid (C16:0) is one of the main features of Neptuniibacter fatty acids, comprising approximately 15–26% of the total (S1 Table). C16:0 is one of the main products of the FAS II pathway, suggesting PT1^T could also produce C16:0 based on genome comparisons (S6 Fig). In Neptunibacter species, C16:1 and C18:1 together make up over 60% of the total fatty acids (S1 Table), so monounsaturated fatty acids are also major features of the fatty acid profile in PT1^T. Monounsaturated fatty acids can be produced through ω7 monounsaturated fatty acid synthesis initiated by isomerization of trans-2-decenoyl-ACP into cis-3-decenoyl-ACP by fabA gene product (S4 Fig). After elongation by fabB gene product, the acyl chain is returned to the FAS II pathway and goes through further elongation, producing C16:1ω7c and C18:1ω7c [5, 6, 36]. The genome comparisons predicted that all strains including PT1^T are capable of producing C16:1ω7c and C18:1ω7c by fabA and fabB gene products. 3-hydroxylated FAs, which are the primary fatty acids in lipid A as well as in ornithine-containing lipids, could be supplied by the FAS II pathway since 3-hydroxy-acyl-ACP is known to be normally intermediated in the FAS II elongation cycle [5, 6, 36]. Since no genes responsible for the synthesis of ornithine-containing lipids were found in genomes of PT1^T or any other species in either genus, it is likely that 3-hydroxylated FAs in PT1^T originate in lipid A [5, 6].

A comparative genome survey of the genes responsible for the FAS II pathway on the PT1^T genome reveals the presence of a core gene set, and genomic structures of FAS II core genes are likely to be retained between described Neptuniibacter species (S5 and S6 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. PT1^T is also potentially capable of making C10:0 3-OH which is commonly found in this genus because of the presence of the lpxA gene. lpxA is responsible for the incorporation of 3-hydroxyacyl to UDP-N-acetyl-α-D-glucosamine, which is a primary reaction to the biosynthesis of lipid A [37].

Comparative analysis among Neptuniibacter species including PT1^T also revealed a complete gene set for the production of PG and PE; plsX, plsY, plsC, cdsA, pssA, psd, pgsA and pgpA (S5 and S6 Figs), which indicate that PT1^T produces PG and PE as major polar lipids, similar to other Neptuniibacter species.

The only respiratory quinone reported from previously described Neptuniibacter species is ubiquinone-8 (Q-8) (S1 Table). Biosynthesis of Q-8 consists of nine steps, and Ubi proteins are involved in each reaction; side chain synthesis by ispB gene product, core biosynthetic pathway by ubiC, ubiA, ubiD, ubiX, ubiI, ubiG, ubiH, ubiE, and ubiF, and accessory hypothetical functions by three genes ubiB, ubiJ, and ubiK (S7 Fig) [5, 6, 38]. The set of genes was identified in PT1^T genome, of which results strongly suggest that the predominant ubiquinone of PT1^T is Q-8 (S7 Fig).

Conclusions

A combination of modern genome taxonomic studies including in silico chemical taxonomy revealed that strain PT1^T is a new species in the genus Neptuniibacter. The name Neptuniibacter victor sp. nov. (PT1^T = JCM 35563^T = LMG 32868^T) is proposed.

Description of Neptuniibacter victor sp. nov.

Neptuniibacter victor (vic’tor. L. masc. n. victor, the winner, referring to the predicted ecophysiology of this bacterium possessing T6SS in sea cucumber aquaria, where the bacterium was isolated).

Gram-negative, motile and aerobic rod. Colonies on Marine Agar (BD) are white and 1.0–3.0 mm in diameter after culture for 3 days. No pigmentation and bioluminescence are observed. Growth occurs at 15°C-30°C. Positive for oxidase. Weakly positive for DNase. Negative for growth on a manually prepare basal seawater medium, catalase, hydrolyses of starch, agar, Tween 20 and 80, and gelatin. The newly described species is characterized by positive for nitrate to nitrite, and utilization of D-glucose, using API20NE. The DNA G+C content is 44.2% and the genome size is 3.95 Mb.

The type strain PT1^T (= JCM 35563^T = LMG 32868^T) was isolated from a pentactula larvae of A. japonicus reared in a laboratory aquarium at Hokkaido University, Hakodate, Japan. The GenBank accession number for the 16S rRNA gene sequence of the type strain is LC716006. The complete genome sequence of the strain is deposited in the DDBJ/ENA/GenBank under the accession number AP025763.

Supporting information

S1 Table. Chemotaxonomic profile of previously described Neptuniibacter.

PG, phosphatidylglycerol; PE, phosphatidylethanolamine; DPG, diphosphatidylglycerol; PL, phospholipids; AL, aminolipid; PN, phosphoaminolipid; nd: not determined.

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S1 Fig. Heat map representation of ANI values.

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S2 Fig. Heat map representation of AAI values.

Reference genomes were retrieved from NCBI database.

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S3 Fig. In silico phenotyping of PT1 by Traitar.

0: negative, 1:phypat positive, 2: phypat+PGL positive, 3: both predictions positive.

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Click here for additional data file.^{(488.3KB, pdf)}

S4 Fig. Predicted fatty acid synthetic pathway in Neptuniibacter.

Predicted fatty acid synthetic pathway in Neptuniibacter. ACP: acyl-carrier protein; accABCD: acetyl-CoA carboxylase complex; fabA: 3-hydroxyacyl-ACP dehydrase/trans-2-decanoyl-ACP isomerase; fabB: 3-ketoacyl-ACP synthase Ⅰ; fabD: malonyl-CoA: ACP transacylase; fabF: 3-ketoacyl-ACP synthase Ⅱ; fabG: 3-ketoacyl-ACP reductase; fabI: enoyl-ACP reductase Ⅰ; fabV: enoyl-ACP reductase; fabY: 3-ketoacyl-ACP synthase; fabZ: 3-hydroxyacyl-ACP dehydratase; lpxA: glucosamine N-acyltransferase.

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S5 Fig. Gene structure of FAS associated genes.

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S6 Fig. Genomic distribution of fab and associated genes.

Protein/enzyme name each gene is coding: fabA: 3-hydroxyacyl-ACP dehydrase/trans-2-decanoyl-ACP isomerase; fabB: 3-ketoacyl-ACP synthase Ⅰ; fabD: malonyl-CoA: ACP transacylase; fabF: 3-ketoacyl-ACP synthase Ⅱ; fabG: 3-ketoacyl-ACP reductase; fabI: enoyl-ACP reductase Ⅰ; fabV: enoyl-ACP reductase; fabY: 3-ketoacyl-ACP synthase; fabZ: 3-hydroxyacyl-ACP dehydratase; acpP: Acyl-carrier protein; plsX: FA/phospholipid synthesis protein; lpxAD: glucosamine N-acyltransferase; lpxB: lipid-A-disaccharide synthase.

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Click here for additional data file.^{(732KB, pdf)}

S7 Fig. Predicted Q-8 synthetic pathways in Neptuniibacter.

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Acknowledgments

We gratefully thank for Professor (Emeritus) Aharon Oren, The Hebrew University of Jerusalem, for his advice on bacterial names.

Data Availability

The GenBank accession number for the 16S rRNA gene sequence of the type strain PT1 is LC716006. The whole genome sequence of the PT1 and Neptuniibacter halophilus LMG 25378T have been deposited to DDBJ/ENA/GenBank under the accession numbers AP025763, and AP027292-AP027293. Raw reads used for those genome assembly have been also deposited at GenBank under accession number DRA015857.

Funding Statement

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.

References

1.Arahal DR, Lekunberri I, Gonzalez JM, Pascual J, Pujalte MJ, Pedros-Alio C, et al. Neptuniibacter caesariensis gen. nov., sp. nov., a novel marine genome-sequenced gammaproteobacterium. Int J Syst Evol Microbiol. 2007; 57:1000–1006. doi: 10.1099/ijs.0.64524-0 [DOI] [PubMed] [Google Scholar]
2.Chen MH, Sheu SY, Chiu TF, Chen WM. Neptuniibacter halophilus sp. nov., isolated from a salt pan, and emended description of the genus Neptuniibacter. Int J Syst Evol Microbiol. 2012; 62:1104–1109. doi: 10.1099/ijs.0.030379-0 [DOI] [PubMed] [Google Scholar]
3.Dieguez AL, Balboa S, Magnesen T, Romalde JL. Neptuniibacter pectenicola sp. nov. and Neptuniibacter marinus sp. nov., two novel species isolated from a Great scallop (Pecten maximus) hatchery in Norway and emended description of the genus Neptuniibacter. Syst App Microbiol. 2017; 40:80–85. doi: 10.1016/j.syapm.2016.12.002 [DOI] [PubMed] [Google Scholar]
4.Yu J, Sakai Y, Mino S, Sawabe T. Characterization of microbiomes associated with the early life stages of sea cucumber Apostichopus japonicus Selenka. Front Mar Sci. 2022; 9, 37. doi: 10.3389/fmars.2022.801344 [DOI] [Google Scholar]
5.Yamano R, Yu J, Jiang C, Haditomo AHC, Mino S, Sakai Y, et al. Taxonomic revision of the genus Amphritea supported by genomic and in silico chemotaxonomic analyses, and the proposal of Aliamphritea gen. nov. PLoS ONE. 2022; 17: e0271174. doi: 10.1371/journal.pone.0271174 [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Yamano R, Yu J, Haditomo AHC, Jiang C, Mino S, Romalde JL, et al. Genome taxonomy of the genus Thalassotalea and proposal of Thalassotalea hakodatensis sp. nov. isolated from sea cucumber larvae. PLoS ONE. 2023; 18: e0286693. doi: 10.1371/journal.pone.0286693 [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Al-Saari N, Gao F, Amin AKMR, Sato K, Sato K, Mino S, et al. Advanced microbial taxonomy combined with genome-based-approaches reveals that Vibrio astriarenae sp. nov., an agarolytic marine bacterium, forms a new clade in Vibrionaceae. PLoS ONE. 2015; 10: e0136279. doi: 10.1371/journal.pone.0136279 [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Amin AKMR, Tanaka M, Al-Saari N, Feng G, Mino S, Ogura Y, et al. Thaumasiovibrio occultus gen. nov. sp. nov. and Thaumasiovibrio subtropicus sp. nov. within the family Vibrionaceae, isolated from coral reef seawater off Ishigaki Island, Japan. Syst Appl Microbiol. 2017; 40:290–296. doi: 10.1016/j.syapm.2017.04.003 [DOI] [PubMed] [Google Scholar]
9.Sawabe T, Ogura Y, Matsumura Y, Feng G, Amin AKMR, Mino S, et al. Updating the Vibrio clades defined by multilocus sequence phylogeny: proposal of eight new clades, and the description of Vibrio tritonius sp. nov. Front Microbiol. 2013; 4:414. doi: 10.3389/fmicb.2013.00414 [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Tanaka M, Hongyu B, Jiang C, Mino S, Meirelles PM, Thompson F, et al. Vibrio taketomensis sp. nov. by genome taxonomy. Syst Appl Microbiol. 2020; 43:126048. doi: 10.1016/j.syapm.2019.126048 [DOI] [PubMed] [Google Scholar]
11.Weimann A, Mooren K, Frank J, Pope PB, Bremges A, McHardy AC. From genomes to phenotypes: traitar, the microbial trait analyzer. mSystems. 2016; 1, e00101–e00116. doi: 10.1128/mSystems.00101-16 [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Wick RR, Judd LM, Gorrie CL, Holt KE. Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput Biol. 2017; 13:e1005595. doi: 10.1371/journal.pcbi.1005595 [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Tanizawa Y, Fujisawa T, Nakamura Y. DFAST: a flexible prokaryotic genome annotation pipeline for faster genome publication. Bioinformatics. 2018; 34:1037–1039. doi: 10.1093/bioinformatics/btx713 [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Mol Biol Evol. 2018; 35:1547–1549. doi: 10.1093/molbev/msy096 [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Stecher G, Tamura K, Kumar S. Molecular Evolutionary Genetics Analysis (MEGA) for macOS. Mol Biol Evol. 2020; 33:1870–1874. doi: 10.1093/molbev/msz312 [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Lee I, Kim YO, Park SC, Chun J. OrthoANI: an improved algorithm and software for calculating average nucleotide identity. Int J Syst Evol Microbiol. 2015; 66:1100–1103. doi: 10.1099/ijsem.0.000760 [DOI] [PubMed] [Google Scholar]
17.Meier-Kolthoff JP, Auch AF, Klenk HP, Göker M. Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinformatics. 2013; 14:60. doi: 10.1186/1471-2105-14-60 [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Rodriguez-R LM, Konstantinidis KT. The enveomics collection: a toolbox for specialized analyses of microbial genomes and metagenomes. PeerJ. 2016; 4:e1900v1. doi: 10.7287/peerj.preprints.1900v1 [DOI] [Google Scholar]
19.Huson D, Bryant D. Application of phylogenetic networks in evolutionary studies. Mol Biol Evol. 2006; 23:254–267. doi: 10.1093/molbev/msj030 [DOI] [PubMed] [Google Scholar]
20.Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, et al. Clustal W and Clustal X version 2.0. Bioinformatics. 2007; 23:2947–2948. doi: 10.1093/bioinformatics/btm404 [DOI] [PubMed]
21.Eren AM, Kiefl E, Shaiber A, Veseli I, Miller SE, Schechter MS. Community-led, integrated, reproducible multi-omics with anvi’o. Nat Microbiol. 2021; 6:3–6. doi: 10.1038/s41564-020-00834-3 [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Delmont TO, Eren AM. Linking pangenomes and metagenomes: the Prochlorococcus metapangenome. PeerJ. 2018; 6:e4320. doi: 10.7717/peerj.4320 [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJE. The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc. 2015; 10:845–858. doi: 10.1038/nprot.2015.053 [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Yarza P, Yilmaz P, Pruesse E. Uniting the classification of cultured and uncultured bacteria and archaea using 16S rRNA gene sequences. Nat Rev Microbiol. 2014; 12:635–645. doi: 10.1038/nrmicro3330 [DOI] [PubMed] [Google Scholar]
25.Kim M, Oh HS, Park SC, Chun J. Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. Int J Syst Evol Microbiol. 2014; 64:346–351. doi: 10.1099/ijs.0.059774-0 [DOI] [PubMed] [Google Scholar]
26.Chun J, Oren A, Ventosa A, Christensen H, Arahal DR, da Costa MS, et al. Proposed minimal standards for the use of genome data for the taxonomy of prokaryotes. Int J Syst Evol Microbiol. 2018; 68:461–466. doi: 10.1099/ijsem.0.002516 [DOI] [PubMed] [Google Scholar]
27.Konstantinidis KT, Tiedje JM. Towards a genome-based taxonomy for prokaryotes. J Bacteriol Res. 2005; 6258–6264. doi: 10.1128/JB.187.18.6258-6264.2005 [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Spearea L, Cecereb AG, Guckesb KR, Smitha S, Wollenbergc MS, Mandeld MJ, et al. Bacterial symbionts use a type VI secretion system to eliminate competitors in their natural host. Proc Natl Aca Sci USA. 2018; 115: E8528–E8537. doi: 10.1073/pnas.1808302115 [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Bongrand C, Ruby EG. The impact of Vibrio fischeri strain variation on host colonization. Curr Opin Microbiol. 2019; 50:15–19. doi: 10.1016/j.mib.2019.09.002 [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Cole JA, Richardson DJ. Respiration of nitrate and nitrite. EcoSal Plus. 3.2.5, doi: 10.1128/ecosal.3.2.5 [DOI] [PubMed] [Google Scholar]
31.Malm S, Tiffert Y, Micklinghoff J, Schultze S, Joost I, Weber I, et al. The roles of the nitrate reductase NarGHJI, the nitrite reductase NirBD and the response regulator GlnR in nitrate assimilation of Mycobacterium tuberculosis. Microbiology. 2009; 155:1332–1339. [DOI] [PubMed] [Google Scholar]
32.Moreno-Vivian C, Cabello P, Martinez-Luque M, Blasco L, Castillo R. Prokaryotic nitrate reduction: molecular properties and functional distinction among bacterial nitrate reductases. J Bacteriol. 181:6573–6584. doi: 10.1128/JB.181.21.6573-6584.1999 [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Wang Y, Ruby EG. The roles of NO in microbial symbioses. Cellular Microbiol. 2011; 13:518–526. doi: 10.1111/j.1462-5822.2011.01576.x [DOI] [PMC free article] [PubMed] [Google Scholar]
34.Yamazaki Y, Meirelles PM, Mino S, Suda W, Oshima K, Hattori M, et al. Individual Apostichopus japonicus fecal microbiome reveals a link with polyhydroxybutyrate producers in host growth gaps. Sci Rep. 2016; 6:21631. doi: 10.1038/srep21631 [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Janßen HJ, Steinbüchel A. Fatty acid synthesis in Escherichia coli and its applications towards the production of fatty acid based biofuels. Biotechnol Biofuels. 2014; 7:7. doi: 10.1186/1754-6834-7-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Lopez-Lara IM, Geiger O. Formation of fatty acids. In: Timmis KN. Handbook of Hydrocarbon and Lipid Microbiology. Springer-Verlag Berlin Heidelberg. 2010; pp. 386–393. doi: 10.1007/978-3-540-77587-4_26 [DOI] [Google Scholar]
37.Coleman J, Raetz CR. First committed step of lipid A biosynthesis in Escherichia coli: sequence of the lpxA gene. J Bacteriol. 1988; 170:1268–74. doi: 10.1128/jb.170.3.1268-1274.1988 [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Abby SS, Kazemzadeh K, Vragniau C, Pelosi L, Perrel F. Advances in bacterial pathways for the biosynthesis of ubiquinone. Biochim Biophys Acta Bioenerg. 2020; 1861:11. doi: 10.1016/j.bbabio.2020.148259 [DOI] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0290060.r001

Decision Letter 0

Karel Sedlar

7 Mar 2023

PONE-D-23-05318Genome taxonomy of the genus Neptuniibacter and proposal of Neptuniibacter victor sp. nov. isolated from sea cucumber larvae.PLOS ONE

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PLoS One. 2023 Aug 15;18(8):e0290060. doi: 10.1371/journal.pone.0290060.r002

Author response to Decision Letter 0

13 Mar 2023

Dear Dr. Sedlar, Academic Editor, PLOS ONE

We gratefully thank to the editor for comments concerning our manuscript entitled “Genome taxonomy of the genus Neptuniibacter and proposal of Neptuniibacter victor sp. nov. isolated from sea cucumber larvae”, PONE-D-23-05318.

We have described accession numbers and these are now publicly available.

Academic Editor: Thank you for submitting your manuscript to PLOS ONE. Before I'll send your manuscript to reviewers, I need to secure that the manuscript meets publication criteria. Since the genome assembly is a part of your manuscript for the sake of reproducibility you need to make all data publicly available. Therefore, you need to upload raw sequencing data to Sequence Read Archive or similar database. Moreover, your accessions for N. halophilus LMG 25378T are not currently available in GenBank/EMBL/DDBJ. Please make all necessary data available for peer review.

Reply: Thank you for your comments. We provide DRA numbers and all sequences are now publicly available.

Attachment

Submitted filename: 13Mar23_response_DRA.docx

Click here for additional data file.^{(63.6KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0290060.r003

Decision Letter 1

Karel Sedlar

6 Jun 2023

PONE-D-23-05318R1

Genome taxonomy of the genus Neptuniibacter and proposal of Neptuniibacter victor sp. nov. isolated from sea cucumber larvae.

PLOS ONE

Dear Dr. Sawabe,

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Kind regards,

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Academic Editor

PLOS ONE

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During our internal evaluation of the manuscript, we found significant text overlap between your submission and your previously published work (In silico chemical taxonomy section in particular):

https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0271174

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Reviewer #1: The article "Genome taxonomy of the genus Neptuniibacter and proposal of Neptuniibacter victor. sp nov. isolated from sea cucumber larvae" provides insights into a newly discovered strain PT1T found in sea cucumber larvae where genotypic and phenotypic in silico analysis is conducted. The article is well-written and provides a clear description of the research findings. However, several points need to be clarified.

Major comments:

The article mentions that the Unicycler tool was used for genome assembly. However, it is not clear if any preprocessing was done on the Illumina data, such as adapter removal, which can significantly influence results. Moreover, I am missing information on whether the quality of the sequencing data was checked.

The author states that whole genome sequences were obtained, and as a result, contigs were obtained (Tab. 1). In the case of more contigs, it is unclear if contigs contain only chromosome sequences or if plasmids are also present in the assembled contigs. If yes, are genes from plasmids included in the pan-genome analysis?

On line 107, the article states that the ML tree was generated from 100 replicates, but Figure 1 indicates that 1000 replicates were performed. This discrepancy should be clarified.

The article references the acceptance of results according to formula 2 for the OGRIs calculation (line 115), but formula 2 is not provided.

While AAI values were calculated for PT1T and other Neptuniibacter species, the obtained values are not provided. It is unclear how the author determined which genomes were used or not for analysis, as indicated in Table 1. The same issue applies to the MLSA column in Table 1.

The article states that phylogenetic network reconstruction was performed for MLSA, but there is no description of the settings used for the SplitsTree tool or the method of construction used.

In the section on pan and core genome analysis, it is not clear if the same tool was used for both analyses.

In the section Pan and core genomes and its ecogenomic interpretation, the author mentions that the assimilatory nitrate reduction pathway was predicted in the analyzed strain. It is unclear how this pathway was predicted and if a specific tool was used.

There are some minor comments regarding the article that I would like to make:

The version number should be included for each tool used in this study (e.g. lines 74, 113, 148 etc.).

The citation for the MEGAX tool is missing (line 105).

The word "Illumina" should start with a capital letter (line 91).

In Tab. 1, there should be some units for the size column.

Citations need to be sorted in ascending order (line 172).

The abbreviation OF is used, but its meaning is not explained (line 72).

Reviewer #2: PONE-D-23-05318R1

The authors present a study: Genome taxonomy of the genus Neptuniibacter and a proposal of Neptuniibacter victor sp. nov. isolated from sea cucumber larvae.

The methods used demonstrably confirm that the described strain PT1T belongs to a new, previously undescribed Neptuniibacter species. The article is supported by sufficient literature references.

The sequencing methods, genome analysis and MLST analysis are described and performed in detail and provide valuable information. On the other hand, my main complaint is that the article provides chemotaxonomic results and phenotyping mainly from the in silico approach. It brings mainly predicted phenotypic characteristics of the studied strain PT1T. I miss in the discussion any mention of the reliability of the analysis with Traitar (Microbial Trait Analyzer) and the used in silico chemical taxonomy and maybe comparison with in vitro methods. In my opinion, a description of genomic and phenotypic traits (tested in vitro) should be more balanced.

I recommend the article for publication with minor revision.

Other comments and recommendations:

Line 127, Table 1: It would be useful to adjust the width of the columns to make the data more readable.

Line 185, Table 2: The numbers of the reference type strains should be indicated.

Table 2: The range of the temperatures tested is too wide (4°C, 10°C, ...37°C). It seems reasonable and logical to test the growth also at 20 or 25°C.

The phenotyping data are rather insufficient for the description of the novel species. I suggest testing the commercial kit API ZYM to complement the phenotypic description. This commercial kit is well standardised and has been used in a species description of other Neptuniibacter spp. Also test a hydrolysis of gelatine and Tween 80 if possible (tests mentioned in previous Neptuniibacter species).

Line 187: Please complete the following sentence, there is something missing in the text: Phenotypic data for Neptuniibacter .... are from this study and .... [1-3].

Line 207: Please complete the following sentence, there is something missing in the text: Fig 3. Anvi'o representation of the pangenome of strain PT1T and Neptuniibacter ...

The word Unique, written in italics, is somewhat confusing together with the species name in Fig. 3. It could be written in brackets.

Please include the reference strain numbers in all tables and figures; both in the main text and in the supplementary data.

Line 310: There is a mistake in the doi number. The correct one is doi:310 10.1099/ijs.0.64524-0.

Please add to the discussion section information on the reliability/confidence of the in silico approaches and assess their suitability for the genera studied.

It would be interesting to compare the predicted properties (Fig. S3) with in vitro tests from previous descriptions of Neptuniibacter spp. Do the results agree? In which cases is the gene not expressed?

Fig S7: There is an extra dot.

**********

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: Yes: Marketa Nykrynova

Reviewer #2: No

**********

PLoS One. 2023 Aug 15;18(8):e0290060. doi: 10.1371/journal.pone.0290060.r004

Author response to Decision Letter 1

1 Jul 2023

Dear Professor Karel Sedlar, the editor, and reviewers,

We appreciate editor and reviewers for constructive suggestions for PONE-D-23-05318R1. We improved the manuscript according to the editorial office and reviewers’ comments. Responses for specific comments are described as follows. All changes were found in the tracking-on file separately uploaded. We also refine Tables 1 and 2, and Figs 1-3. Table 1 was not framed in body, so we also attached table1.tif as other material.

Editorial office

1. During our internal evaluation of the manuscript, we found significant text overlap between your submission and your previously published work (In silico chemical taxonomy section in particular)

Response: Thank you for the suggestion. We used almost same approach of previously published papers, so many parts of body text were overlapped. However, we did remove redundancy and put citations of previous paper.

Reviewer: 1

1. The article mentions that the Unicycler tool was used for genome assembly. However, it is not clear if any preprocessing was done on the Illumina data, such as adapter removal, which can significantly influence results. Moreover, I am missing information on whether the quality of the sequencing data was checked.

Response: Thank you so much for the comments after careful reading. We did not perform preprocessing Illumina reads at this time because we confirmed QV>27 and no adaptor and barcode sequences of the Illumina read using the FastQC program before the assembly. We put the explanation in body.

2. For pangenome analyses;

1) The author states that whole genome sequences were obtained, and as a result, contigs were obtained (Tab. 1). In the case of more contigs, it is unclear if contigs contain only chromosome sequences or if plasmids are also present in the assembled contigs. If yes, are genes from plasmids included in the pan-genome analysis?

2) In the section on pan and core genome analysis, it is not clear if the same tool was used for both analyses.

3) In the section Pan and core genomes and its ecogenomic interpretation, the author mentions that the assimilatory nitrate reduction pathway was predicted in the analyzed strain. It is unclear how this pathway was predicted and if a specific tool was used.

Response:

1) Thank you so much for the comments. Actually, pan-genome analyses only use genomes, but unfortunately, we did not obtain complete genome sequences ourselves from the other three Neptuniibacter type strains. Due to being not survived during shipment from European collections by the heat wave in Europe in 2022 (too hot summer during shipments), we were unable to obtain those type strains. So, we decided to use draft genomes obtained from public databases. These sequences might include plasmids, but we could not select and differentiate them. PT1 has no plasmid, but N. halophilus has one plasmid.

2) We used Anvio for both pan- and core- analyses. We modified body in related parts.

3) For assimilatory nitrate reduction, we evaluated only by presence of nasAC and nirA genes unique in PT1. We added one more reference for that.

3. For the other methodology

1) On line 107, the article states that the ML tree was generated from 100 replicates, but Figure 1 indicates that 1000 replicates were performed. This discrepancy should be clarified.

2) The article references the acceptance of results according to formula 2 for the OGRIs calculation (line 115), but formula 2 is not provided.

3) While AAI values were calculated for PT1T and other Neptuniibacter species, the obtained values are not provided.

4) It is unclear how the author determined which genomes were used or not for analysis, as indicated in Table 1. The same issue applies to the MLSA column in Table 1.

5) The article states that phylogenetic network reconstruction was performed for MLSA, but there is no description of the settings used for the SplitsTree tool or the method of construction used.

Response: Thank you for your comments. All points were fixed and/or corrected accordingly. Rerated to 1) and 5), we refined 16S and MLSA network as well, in particular, regions used for those phylogenetic analyses were described. For 2), we modified the body to cover formula 2 explanation by ref 17, in which it is explained that Formula 2 is optimized for using incomplete genomes. We both analyzed complete and incomplete genomes, so we selected formula 2. AAI values were already provided in body around L170 and S2 Fig, but improved the body. To be clear which genome we determined ourselves, we put “in this study” in Table 1. For MLSA, “concatenation of sequences and phylogenetic NeighborNet reconstruction were performed using SplitsTree 4.16.2 with options of Jukes-Cantor correction, gap-exclusion and 1,000 bootstrap”, but eliminating redundancy of previously published paper (Yamano et al., 2023, PLoS One), we add this ref.

4. There are some minor comments regarding the article that I would like to make:

1) The version number should be included for each tool used in this study (e.g. lines 74, 113, 148 etc.).

2) The citation for the MEGAX tool is missing (line 105).

3) The word "Illumina" should start with a capital letter (line 91).

4) In Tab. 1, there should be some units for the size column.

5) Citations need to be sorted in ascending order (line 172).

6) The abbreviation OF is used, but its meaning is not explained (line 72).

Response: Thank you for your comments. All points were fixed and/or corrected accordingly.

Reviewer: 2

1. The authors present a study: Genome taxonomy of the genus Neptuniibacter and a proposal of Neptuniibacter victor sp. nov. isolated from sea cucumber larvae. The methods used demonstrably confirm that the described strain PT1T belongs to a new, previously undescribed Neptuniibacter species. The article is supported by sufficient literature references. The sequencing methods, genome analysis and MLST analysis are described and performed in detail and provide valuable information. On the other hand, my main complaint is that the article provides chemotaxonomic results and phenotyping mainly from the in silico approach. It brings mainly predicted phenotypic characteristics of the studied strain PT1T. I miss in the discussion any mention of the reliability of the analysis with Traitar (Microbial Trait Analyzer) and the used in silico chemical taxonomy and maybe comparison with in vitro methods. In my opinion, a description of genomic and phenotypic traits (tested in vitro) should be more balanced.

2. Other comments and recommendations:

1) Line 127, Table 1: It would be useful to adjust the width of the columns to make the data more readable.

2) Line 185, Table 2: The numbers of the reference type strains should be indicated.

Response: Corrected accordingly.

3) Table 2: The range of the temperatures tested is too wide (4°C, 10°C, ...37°C). It seems reasonable and logical to test the growth also at 20 or 25°C.

Response: We tested 4, 10, 15, 20, 25, 30, 37°C. As indicated in footnote, all strains grow at 15-30°C.

4) The phenotyping data are rather insufficient for the description of the novel species. I suggest testing the commercial kit API ZYM to complement the phenotypic description. This commercial kit is well standardised and has been used in a species description of other Neptuniibacter spp. Also test a hydrolysis of gelatine and Tween 80 if possible (tests mentioned in previous Neptuniibacter species).

Response: According to the comments, we did perform API20NE, but not APIzym due to no distribution currently in Japan. Tween 80 test was also performed, and described the negative results in body. Traitar prediction results were removed from the description section.

5) Line 187: Please complete the following sentence, there is something missing in the text: Phenotypic data for Neptuniibacter .... are from this study and .... [1-3].

Response: Corrected accordingly.

6) Line 207: Please complete the following sentence, there is something missing in the text: Fig 3. Anvi'o representation of the pangenome of strain PT1T and Neptuniibacter ... The word Unique, written in italics, is somewhat confusing together with the species name in Fig. 3. It could be written in brackets.

Response: Changed accordingly.

7) Please include the reference strain numbers in all tables and figures; both in the main text and in the supplementary data.

8) Line 310: There is a mistake in the doi number. The correct one is doi:310 10.1099/ijs.0.64524-0.

Response: Changed accordingly.

9) Please add to the discussion section information on the reliability/confidence of the in silico approaches and assess their suitability for the genera studied.

Response: Thank you so much for the comments. We did compared results by Traitar and experiments, and 82% accuracy in average was evaluated. We did add some results and sentences in related section. We also attached this result here (please see attached file separately). Finally, we did observe some growth in API20NE system, we did remove the Traitar prediction results from the description.

Fig S7: There is an extra dot.

Response: Changed accordingly.

Attachment

Submitted filename: Response_letter_revision_1July23.docx

Click here for additional data file.^{(37.8KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0290060.r005

Decision Letter 2

Karel Sedlar

24 Jul 2023

PONE-D-23-05318R2Genome taxonomy of the genus Neptuniibacter and proposal of Neptuniibacter victor sp. nov. isolated from sea cucumber larvae.PLOS ONE

Dear Dr. Sawabe,

While I am overall happy with changes you made and I really appreciate you did additional experiment, reviewer 1 still found several issues with your manuscript. These are mostly grammar mistakes and small technical issues. Nevertheless, PLOS ONE is a respected journal which guarantees high technical quality of published articles. Therefore, I am forced to ask you for an additional round of revisions. Please be sure you adressed all issues that reviewer had raised and re-read your manuscript carefully to prevent yourselves from introducing any other mistakes. I believe you can address these issues in a very short time so your manuscript could be finally accepted for publication. Please submit your revised manuscript by Sep 04 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

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'.

We look forward to receiving your revised manuscript.

Kind regards,

Karel Sedlar, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

Additional Staff Editor Comments:

Please address the text overlap concern we identified between your submission and your previously published work (In silico chemical taxonomy in the Result section in particular):

https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0271174

Please revise the manuscript to rephrase the duplicated text and fully cite all your sources, where appropriate.

We will carefully review your manuscript upon resubmission, so please ensure that your revision is thorough.

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

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: (No Response)

**********

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

Reviewer #1: Yes

**********

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

Reviewer #1: Yes

**********

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

Reviewer #1: Yes

**********

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

Reviewer #1: Yes

**********

6. Review Comments to the Author

Reviewer #1: For molecular phylogenetic analysis:

The authors added information about the settings used for MLSA (line 125). However, on line 131, they mentioned that "almost same setting" was used. I am missing information on how the setting was modified.

Other comments:

1) Line 28: There is an unnecessary gap between the range of numbers (72.8%- 74.8%).

2) Line 70: In the reference to citations, there are 2 hyphens instead of one.

3) Line 94: I guess QV means quality value, but in the text, it is written as quality vale. The word "Illumina" is missing the letter "m".

4) Line 98: There should be "assembly" instead of "assemble".

5) Line 120: The shortcut DDH is not explained.

6) Line 142: In the word "N. 10aesariensis" the number 10 should be removed.

7) Line 174: There should be "were" instead of "was".

8) Regarding the tools, the referencing of versions should be standardized. Sometimes it is written as "ver.," and other times as "v."

**********

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: Yes: Markéta Nykrýnová

**********

PLoS One. 2023 Aug 15;18(8):e0290060. doi: 10.1371/journal.pone.0290060.r006

Author response to Decision Letter 2

25 Jul 2023

Dear Professor Karel Sedlar, the editor, and reviewers,

We appreciate editor and reviewers for constructive suggestions for PONE-D-23-05318R2. We improved the manuscript according to the editorial office and reviewers’ comments. Responses for specific comments are described as follows. All changes were found in the tracking-on file separately uploaded. Again, Table 1 was not framed in body, so we also attached table1.tif as other material.

Editorial office

1. Please address the text overlap concern we identified between your submission and your previously published work (In silico chemical taxonomy in the Result section in particular):

https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0271174. We would like to make you aware that copying extracts from previous publications, especially outside the methods section, word-for-word is unacceptable. In addition, the reproduction of text from published reports has implications for the copyright that may apply to the publications. Please revise the manuscript to rephrase the duplicated text and fully cite all your sources, where appropriate.

We will carefully review your manuscript upon resubmission, so please ensure that your revision is thorough.

Response: Thank you for the suggestion. Once again, we used almost same approach of previously published papers, so many parts of body text were overlapped. However, we did rewrite, remove redundancy, and add reference citations. Reference format was corrected accordingly.

Reviewer: 1

1. For molecular phylogenetic analysis:

Response: Thank you so much for the comments. Related part was improved, in particular, line 125 is fused to the first sentence of this section in removing redundancy.

Other comments:

1) Line 28: There is an unnecessary gap between the range of numbers (72.8%- 74.8%).

2) Line 70: In the reference to citations, there are 2 hyphens instead of one.

3) Line 94: I guess QV means quality value, but in the text, it is written as quality vale. The word "Illumina" is missing the letter "m".

4) Line 98: There should be "assembly" instead of "assemble".

5) Line 120: The shortcut DDH is not explained.

6) Line 142: In the word "N. 10aesariensis" the number 10 should be removed.

7) Line 174: There should be "were" instead of "was".

8) Regarding the tools, the referencing of versions should be standardized. Sometimes it is written as "ver.," and other times as "v."

Response: Thank you for the suggestion. All corrected accordingly.

Attachment

Submitted filename: Response_letter_3rd_revision_25July23.docx

Click here for additional data file.^{(18.8KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0290060.r007

Decision Letter 3

Karel Sedlar

2 Aug 2023

Genome taxonomy of the genus Neptuniibacter and proposal of Neptuniibacter victor sp. nov. isolated from sea cucumber larvae.

PONE-D-23-05318R3

Dear Dr. Sawabe,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Karel Sedlar, Ph.D.

Academic Editor

PLOS ONE

Additional Staff Editor Comments:

Please address the text overlap concern we identified between your submission and your previously published work (In silico chemical taxonomy in the Result section, in particular lines 259-290):

https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0271174

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0290060.r008

Acceptance letter

Karel Sedlar

4 Aug 2023

PONE-D-23-05318R3

Genome taxonomy of the genus Neptuniibacter and proposal of Neptuniibacter victor sp. nov. isolated from sea cucumber larvae

Dear Dr. Sawabe:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Karel Sedlar

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Chemotaxonomic profile of previously described Neptuniibacter.

PG, phosphatidylglycerol; PE, phosphatidylethanolamine; DPG, diphosphatidylglycerol; PL, phospholipids; AL, aminolipid; PN, phosphoaminolipid; nd: not determined.

(PDF)

Click here for additional data file.^{(173.2KB, pdf)}

S1 Fig. Heat map representation of ANI values.

(PDF)

Click here for additional data file.^{(129.7KB, pdf)}

S2 Fig. Heat map representation of AAI values.

Reference genomes were retrieved from NCBI database.

(PDF)

Click here for additional data file.^{(120KB, pdf)}

S3 Fig. In silico phenotyping of PT1 by Traitar.

0: negative, 1:phypat positive, 2: phypat+PGL positive, 3: both predictions positive.

(PDF)

Click here for additional data file.^{(488.3KB, pdf)}

S4 Fig. Predicted fatty acid synthetic pathway in Neptuniibacter.

(PDF)

Click here for additional data file.^{(358.8KB, pdf)}

S5 Fig. Gene structure of FAS associated genes.

(PDF)

Click here for additional data file.^{(694.3KB, pdf)}

S6 Fig. Genomic distribution of fab and associated genes.

(PDF)

Click here for additional data file.^{(732KB, pdf)}

S7 Fig. Predicted Q-8 synthetic pathways in Neptuniibacter.

(PDF)

Click here for additional data file.^{(289.6KB, pdf)}

Attachment

Submitted filename: 13Mar23_response_DRA.docx

Click here for additional data file.^{(63.6KB, docx)}

Attachment

Submitted filename: Response_letter_revision_1July23.docx

Click here for additional data file.^{(37.8KB, docx)}

Attachment

Submitted filename: Response_letter_3rd_revision_25July23.docx

Click here for additional data file.^{(18.8KB, docx)}

Data Availability Statement

[pone.0290060.ref001] 1.Arahal DR, Lekunberri I, Gonzalez JM, Pascual J, Pujalte MJ, Pedros-Alio C, et al. Neptuniibacter caesariensis gen. nov., sp. nov., a novel marine genome-sequenced gammaproteobacterium. Int J Syst Evol Microbiol. 2007; 57:1000–1006. doi: 10.1099/ijs.0.64524-0 [DOI] [PubMed] [Google Scholar]

[pone.0290060.ref002] 2.Chen MH, Sheu SY, Chiu TF, Chen WM. Neptuniibacter halophilus sp. nov., isolated from a salt pan, and emended description of the genus Neptuniibacter. Int J Syst Evol Microbiol. 2012; 62:1104–1109. doi: 10.1099/ijs.0.030379-0 [DOI] [PubMed] [Google Scholar]

[pone.0290060.ref003] 3.Dieguez AL, Balboa S, Magnesen T, Romalde JL. Neptuniibacter pectenicola sp. nov. and Neptuniibacter marinus sp. nov., two novel species isolated from a Great scallop (Pecten maximus) hatchery in Norway and emended description of the genus Neptuniibacter. Syst App Microbiol. 2017; 40:80–85. doi: 10.1016/j.syapm.2016.12.002 [DOI] [PubMed] [Google Scholar]

[pone.0290060.ref004] 4.Yu J, Sakai Y, Mino S, Sawabe T. Characterization of microbiomes associated with the early life stages of sea cucumber Apostichopus japonicus Selenka. Front Mar Sci. 2022; 9, 37. doi: 10.3389/fmars.2022.801344 [DOI] [Google Scholar]

[pone.0290060.ref005] 5.Yamano R, Yu J, Jiang C, Haditomo AHC, Mino S, Sakai Y, et al. Taxonomic revision of the genus Amphritea supported by genomic and in silico chemotaxonomic analyses, and the proposal of Aliamphritea gen. nov. PLoS ONE. 2022; 17: e0271174. doi: 10.1371/journal.pone.0271174 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0290060.ref006] 6.Yamano R, Yu J, Haditomo AHC, Jiang C, Mino S, Romalde JL, et al. Genome taxonomy of the genus Thalassotalea and proposal of Thalassotalea hakodatensis sp. nov. isolated from sea cucumber larvae. PLoS ONE. 2023; 18: e0286693. doi: 10.1371/journal.pone.0286693 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0290060.ref007] 7.Al-Saari N, Gao F, Amin AKMR, Sato K, Sato K, Mino S, et al. Advanced microbial taxonomy combined with genome-based-approaches reveals that Vibrio astriarenae sp. nov., an agarolytic marine bacterium, forms a new clade in Vibrionaceae. PLoS ONE. 2015; 10: e0136279. doi: 10.1371/journal.pone.0136279 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0290060.ref008] 8.Amin AKMR, Tanaka M, Al-Saari N, Feng G, Mino S, Ogura Y, et al. Thaumasiovibrio occultus gen. nov. sp. nov. and Thaumasiovibrio subtropicus sp. nov. within the family Vibrionaceae, isolated from coral reef seawater off Ishigaki Island, Japan. Syst Appl Microbiol. 2017; 40:290–296. doi: 10.1016/j.syapm.2017.04.003 [DOI] [PubMed] [Google Scholar]

[pone.0290060.ref009] 9.Sawabe T, Ogura Y, Matsumura Y, Feng G, Amin AKMR, Mino S, et al. Updating the Vibrio clades defined by multilocus sequence phylogeny: proposal of eight new clades, and the description of Vibrio tritonius sp. nov. Front Microbiol. 2013; 4:414. doi: 10.3389/fmicb.2013.00414 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0290060.ref010] 10.Tanaka M, Hongyu B, Jiang C, Mino S, Meirelles PM, Thompson F, et al. Vibrio taketomensis sp. nov. by genome taxonomy. Syst Appl Microbiol. 2020; 43:126048. doi: 10.1016/j.syapm.2019.126048 [DOI] [PubMed] [Google Scholar]

[pone.0290060.ref011] 11.Weimann A, Mooren K, Frank J, Pope PB, Bremges A, McHardy AC. From genomes to phenotypes: traitar, the microbial trait analyzer. mSystems. 2016; 1, e00101–e00116. doi: 10.1128/mSystems.00101-16 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0290060.ref012] 12.Wick RR, Judd LM, Gorrie CL, Holt KE. Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput Biol. 2017; 13:e1005595. doi: 10.1371/journal.pcbi.1005595 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0290060.ref013] 13.Tanizawa Y, Fujisawa T, Nakamura Y. DFAST: a flexible prokaryotic genome annotation pipeline for faster genome publication. Bioinformatics. 2018; 34:1037–1039. doi: 10.1093/bioinformatics/btx713 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0290060.ref014] 14.Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Mol Biol Evol. 2018; 35:1547–1549. doi: 10.1093/molbev/msy096 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0290060.ref015] 15.Stecher G, Tamura K, Kumar S. Molecular Evolutionary Genetics Analysis (MEGA) for macOS. Mol Biol Evol. 2020; 33:1870–1874. doi: 10.1093/molbev/msz312 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0290060.ref016] 16.Lee I, Kim YO, Park SC, Chun J. OrthoANI: an improved algorithm and software for calculating average nucleotide identity. Int J Syst Evol Microbiol. 2015; 66:1100–1103. doi: 10.1099/ijsem.0.000760 [DOI] [PubMed] [Google Scholar]

[pone.0290060.ref017] 17.Meier-Kolthoff JP, Auch AF, Klenk HP, Göker M. Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinformatics. 2013; 14:60. doi: 10.1186/1471-2105-14-60 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0290060.ref018] 18.Rodriguez-R LM, Konstantinidis KT. The enveomics collection: a toolbox for specialized analyses of microbial genomes and metagenomes. PeerJ. 2016; 4:e1900v1. doi: 10.7287/peerj.preprints.1900v1 [DOI] [Google Scholar]

[pone.0290060.ref019] 19.Huson D, Bryant D. Application of phylogenetic networks in evolutionary studies. Mol Biol Evol. 2006; 23:254–267. doi: 10.1093/molbev/msj030 [DOI] [PubMed] [Google Scholar]

[pone.0290060.ref020] 20.Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, et al. Clustal W and Clustal X version 2.0. Bioinformatics. 2007; 23:2947–2948. doi: 10.1093/bioinformatics/btm404 [DOI] [PubMed]

[pone.0290060.ref021] 21.Eren AM, Kiefl E, Shaiber A, Veseli I, Miller SE, Schechter MS. Community-led, integrated, reproducible multi-omics with anvi’o. Nat Microbiol. 2021; 6:3–6. doi: 10.1038/s41564-020-00834-3 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0290060.ref022] 22.Delmont TO, Eren AM. Linking pangenomes and metagenomes: the Prochlorococcus metapangenome. PeerJ. 2018; 6:e4320. doi: 10.7717/peerj.4320 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0290060.ref023] 23.Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJE. The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc. 2015; 10:845–858. doi: 10.1038/nprot.2015.053 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0290060.ref024] 24.Yarza P, Yilmaz P, Pruesse E. Uniting the classification of cultured and uncultured bacteria and archaea using 16S rRNA gene sequences. Nat Rev Microbiol. 2014; 12:635–645. doi: 10.1038/nrmicro3330 [DOI] [PubMed] [Google Scholar]

[pone.0290060.ref025] 25.Kim M, Oh HS, Park SC, Chun J. Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. Int J Syst Evol Microbiol. 2014; 64:346–351. doi: 10.1099/ijs.0.059774-0 [DOI] [PubMed] [Google Scholar]

[pone.0290060.ref026] 26.Chun J, Oren A, Ventosa A, Christensen H, Arahal DR, da Costa MS, et al. Proposed minimal standards for the use of genome data for the taxonomy of prokaryotes. Int J Syst Evol Microbiol. 2018; 68:461–466. doi: 10.1099/ijsem.0.002516 [DOI] [PubMed] [Google Scholar]

[pone.0290060.ref027] 27.Konstantinidis KT, Tiedje JM. Towards a genome-based taxonomy for prokaryotes. J Bacteriol Res. 2005; 6258–6264. doi: 10.1128/JB.187.18.6258-6264.2005 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0290060.ref028] 28.Spearea L, Cecereb AG, Guckesb KR, Smitha S, Wollenbergc MS, Mandeld MJ, et al. Bacterial symbionts use a type VI secretion system to eliminate competitors in their natural host. Proc Natl Aca Sci USA. 2018; 115: E8528–E8537. doi: 10.1073/pnas.1808302115 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0290060.ref029] 29.Bongrand C, Ruby EG. The impact of Vibrio fischeri strain variation on host colonization. Curr Opin Microbiol. 2019; 50:15–19. doi: 10.1016/j.mib.2019.09.002 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0290060.ref030] 30.Cole JA, Richardson DJ. Respiration of nitrate and nitrite. EcoSal Plus. 3.2.5, doi: 10.1128/ecosal.3.2.5 [DOI] [PubMed] [Google Scholar]

[pone.0290060.ref031] 31.Malm S, Tiffert Y, Micklinghoff J, Schultze S, Joost I, Weber I, et al. The roles of the nitrate reductase NarGHJI, the nitrite reductase NirBD and the response regulator GlnR in nitrate assimilation of Mycobacterium tuberculosis. Microbiology. 2009; 155:1332–1339. [DOI] [PubMed] [Google Scholar]

[pone.0290060.ref032] 32.Moreno-Vivian C, Cabello P, Martinez-Luque M, Blasco L, Castillo R. Prokaryotic nitrate reduction: molecular properties and functional distinction among bacterial nitrate reductases. J Bacteriol. 181:6573–6584. doi: 10.1128/JB.181.21.6573-6584.1999 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0290060.ref033] 33.Wang Y, Ruby EG. The roles of NO in microbial symbioses. Cellular Microbiol. 2011; 13:518–526. doi: 10.1111/j.1462-5822.2011.01576.x [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0290060.ref034] 34.Yamazaki Y, Meirelles PM, Mino S, Suda W, Oshima K, Hattori M, et al. Individual Apostichopus japonicus fecal microbiome reveals a link with polyhydroxybutyrate producers in host growth gaps. Sci Rep. 2016; 6:21631. doi: 10.1038/srep21631 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0290060.ref035] 35.Janßen HJ, Steinbüchel A. Fatty acid synthesis in Escherichia coli and its applications towards the production of fatty acid based biofuels. Biotechnol Biofuels. 2014; 7:7. doi: 10.1186/1754-6834-7-7 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0290060.ref036] 36.Lopez-Lara IM, Geiger O. Formation of fatty acids. In: Timmis KN. Handbook of Hydrocarbon and Lipid Microbiology. Springer-Verlag Berlin Heidelberg. 2010; pp. 386–393. doi: 10.1007/978-3-540-77587-4_26 [DOI] [Google Scholar]

[pone.0290060.ref037] 37.Coleman J, Raetz CR. First committed step of lipid A biosynthesis in Escherichia coli: sequence of the lpxA gene. J Bacteriol. 1988; 170:1268–74. doi: 10.1128/jb.170.3.1268-1274.1988 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0290060.ref038] 38.Abby SS, Kazemzadeh K, Vragniau C, Pelosi L, Perrel F. Advances in bacterial pathways for the biosynthesis of ubiquinone. Biochim Biophys Acta Bioenerg. 2020; 1861:11. doi: 10.1016/j.bbabio.2020.148259 [DOI] [PubMed] [Google Scholar]

PERMALINK

Genome taxonomy of the genus Neptuniibacter and proposal of Neptuniibacter victor sp. nov. isolated from sea cucumber larvae

Rika Kudo

Ryota Yamano

Juanwen Yu

Shotaro Koike

Alfabetian Harjuno Condro Haditomo

Mayanne A M de Freitas

Jiro Tsuchiya

Sayaka Mino

Fabiano Thompson

Jesús L Romalde

Hisae Kasai

Yuichi Sakai

Tomoo Sawabe

Roles

Abstract

Introduction

Materials and methods

Bacterial strains and phenotyping

Whole genome sequencing

Molecular phylogenetic analysis based on 16S rRNA gene nucleotide sequences

Overall genome relatedness indices (OGRIs)

Table 1. List of genomes used in this study.

Multilocus sequence analysis (MLSA)

Fig 2. MLSA network of PT1T and related Oceanospirillaceae.

Pan and core genome analyses

In silico chemical taxonomy

Results and discussion

Molecular phylogenetic analysis based on 16S rRNA gene nucleotide sequences

Fig 1. ML tree based on 16S rRNA gene nucleotide sequences of strain PT1T and related type strains.

Genomic features and overall genome relatedness indices (OGRIs)

Multilocus sequence analysis (MLSA)

Experimental phenotypic characterization and in silico phenotyping

Table 2. Phenotypic characteristics of PT1T and Neptuniibacter strains.

Pan and core genomes and its ecogenomic interpretation

Fig 3. Anvi’o representation of the pangenome of strain PT1T and Neptuniibacter.

In silico chemical taxonomy

Conclusions

Description of Neptuniibacter victor sp. nov.

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Karel Sedlar

Roles

Author response to Decision Letter 0

Decision Letter 1

Karel Sedlar

Roles

Author response to Decision Letter 1

Decision Letter 2

Karel Sedlar

Roles

Author response to Decision Letter 2

Decision Letter 3

Karel Sedlar

Roles

Acceptance letter

Karel Sedlar

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases

Fig 2. MLSA network of PT1^T and related Oceanospirillaceae.

Fig 1. ML tree based on 16S rRNA gene nucleotide sequences of strain PT1^T and related type strains.

Table 2. Phenotypic characteristics of PT1^T and Neptuniibacter strains.

Fig 3. Anvi’o representation of the pangenome of strain PT1^T and Neptuniibacter.