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

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).

Citation: Kudo R, Yamano R, Yu J, Koike S, Haditomo AHC, de Freitas MAM, et al. (2023) Genome taxonomy of the genus Neptuniibacter and proposal of Neptuniibacter victor sp. nov. isolated from sea cucumber larvae. PLoS ONE 18(8): e0290060. https://doi.org/10.1371/journal.pone.0290060

Editor: Karel Sedlar, Brno University of Technology: Vysoke uceni technicke v Brne, CZECH REPUBLIC

Received: February 23, 2023; Accepted: July 26, 2023; Published: August 15, 2023

Copyright: © 2023 Kudo 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 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: 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.

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].

Download:

Table 1. List of genomes used in this study.

https://doi.org/10.1371/journal.pone.0290060.t001

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.

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).

Download:

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

https://doi.org/10.1371/journal.pone.0290060.g001

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).

Download:

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

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.

https://doi.org/10.1371/journal.pone.0290060.g002

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).

Download:

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

https://doi.org/10.1371/journal.pone.0290060.t002

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.

Download:

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

Layers represent each genome, and the darker areas represent the occurrence of gene clusters. The outer colored bars indicate the “Core” or “Unique” bins.

https://doi.org/10.1371/journal.pone.0290060.g003

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.

https://doi.org/10.1371/journal.pone.0290060.s001

(PDF)

S1 Fig. Heat map representation of ANI values.

https://doi.org/10.1371/journal.pone.0290060.s002

(PDF)

S2 Fig. Heat map representation of AAI values.

Reference genomes were retrieved from NCBI database.

https://doi.org/10.1371/journal.pone.0290060.s003

(PDF)

S3 Fig. In silico phenotyping of PT1 by Traitar.

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

https://doi.org/10.1371/journal.pone.0290060.s004

(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.

https://doi.org/10.1371/journal.pone.0290060.s005

(PDF)

S5 Fig. Gene structure of FAS associated genes.

https://doi.org/10.1371/journal.pone.0290060.s006

(PDF)

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.

https://doi.org/10.1371/journal.pone.0290060.s007

(PDF)

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

https://doi.org/10.1371/journal.pone.0290060.s008

(PDF)

Acknowledgments

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

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.
- View Article
- 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.
- View Article
- 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. pmid:28040300
- View Article
- PubMed/NCBI
- 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.
- View Article
- 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. pmid:35947547
- View Article
- PubMed/NCBI
- 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. pmid:37267301
- View Article
- PubMed/NCBI
- 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. pmid:26313925
- View Article
- PubMed/NCBI
- 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. pmid:28648725
- View Article
- PubMed/NCBI
- 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. pmid:24409173
- View Article
- PubMed/NCBI
- 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. pmid:31862126
- View Article
- PubMed/NCBI
- 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. pmid:28066816
- View Article
- PubMed/NCBI
- 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. pmid:28594827
- View Article
- PubMed/NCBI
- 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. pmid:29106469
- View Article
- PubMed/NCBI
- 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. pmid:29722887
- View Article
- PubMed/NCBI
- Google Scholar
15. Stecher G, Tamura K, Kumar S. Molecular Evolutionary Genetics Analysis (MEGA) for macOS. Mol Biol Evol. 2020; 33:1870–1874. pmid:31904846
- View Article
- PubMed/NCBI
- 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. pmid:26585518
- View Article
- PubMed/NCBI
- 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. pmid:23432962
- View Article
- PubMed/NCBI
- 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.
- View Article
- Google Scholar
19. Huson D, Bryant D. Application of phylogenetic networks in evolutionary studies. Mol Biol Evol. 2006; 23:254–267. pmid:16221896
- View Article
- PubMed/NCBI
- 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. https://doi.org/10.1093/bioinformatics/btm404
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. pmid:33349678
- View Article
- PubMed/NCBI
- Google Scholar
22. Delmont TO, Eren AM. Linking pangenomes and metagenomes: the Prochlorococcus metapangenome. PeerJ. 2018; 6:e4320. pmid:29423345
- View Article
- PubMed/NCBI
- 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. pmid:25950237
- View Article
- PubMed/NCBI
- 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. pmid:25118885
- View Article
- PubMed/NCBI
- 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. pmid:24505072
- View Article
- PubMed/NCBI
- 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. pmid:29292687
- View Article
- PubMed/NCBI
- Google Scholar
27. Konstantinidis KT, Tiedje JM. Towards a genome-based taxonomy for prokaryotes. J Bacteriol Res. 2005; 6258–6264. pmid:16159757
- View Article
- PubMed/NCBI
- 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. pmid:30127013
- View Article
- PubMed/NCBI
- Google Scholar
29. Bongrand C, Ruby EG. The impact of Vibrio fischeri strain variation on host colonization. Curr Opin Microbiol. 2019; 50:15–19. pmid:31593868
- View Article
- PubMed/NCBI
- Google Scholar
30. Cole JA, Richardson DJ. Respiration of nitrate and nitrite. EcoSal Plus. 3.2.5, pmid:26443731
- View Article
- PubMed/NCBI
- 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.
- View Article
- 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. pmid:10542156
- View Article
- PubMed/NCBI
- Google Scholar
33. Wang Y, Ruby EG. The roles of NO in microbial symbioses. Cellular Microbiol. 2011; 13:518–526. pmid:21338463
- View Article
- PubMed/NCBI
- 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. pmid:26905381
- View Article
- PubMed/NCBI
- 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. pmid:24405789
- View Article
- PubMed/NCBI
- 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.
- View Article
- 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.
- View Article
- 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. pmid:32663475
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 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.
View Article
Google Scholar

[2] View Article

[3] Google Scholar

[ref2] 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.
View Article
Google Scholar

[5] View Article

[6] Google Scholar

[ref3] 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. pmid:28040300
View Article
PubMed/NCBI
Google Scholar

[8] View Article

[9] PubMed/NCBI

[10] Google Scholar

[ref4] 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.
View Article
Google Scholar

[12] View Article

[13] Google Scholar

[ref5] 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. pmid:35947547
View Article
PubMed/NCBI
Google Scholar

[15] View Article

[16] PubMed/NCBI

[17] Google Scholar

[ref6] 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. pmid:37267301
View Article
PubMed/NCBI
Google Scholar

[19] View Article

[20] PubMed/NCBI

[21] Google Scholar

[ref7] 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. pmid:26313925
View Article
PubMed/NCBI
Google Scholar

[23] View Article

[24] PubMed/NCBI

[25] Google Scholar

[ref8] 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. pmid:28648725
View Article
PubMed/NCBI
Google Scholar

[27] View Article

[28] PubMed/NCBI

[29] Google Scholar

[ref9] 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. pmid:24409173
View Article
PubMed/NCBI
Google Scholar

[31] View Article

[32] PubMed/NCBI

[33] Google Scholar

[ref10] 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. pmid:31862126
View Article
PubMed/NCBI
Google Scholar

[35] View Article

[36] PubMed/NCBI

[37] Google Scholar

[ref11] 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. pmid:28066816
View Article
PubMed/NCBI
Google Scholar

[39] View Article

[40] PubMed/NCBI

[41] Google Scholar

[ref12] 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. pmid:28594827
View Article
PubMed/NCBI
Google Scholar

[43] View Article

[44] PubMed/NCBI

[45] Google Scholar

[ref13] 13. Tanizawa Y, Fujisawa T, Nakamura Y. DFAST: a flexible prokaryotic genome annotation pipeline for faster genome publication. Bioinformatics. 2018; 34:1037–1039. pmid:29106469
View Article
PubMed/NCBI
Google Scholar

[47] View Article

[48] PubMed/NCBI

[49] Google Scholar

[ref14] 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. pmid:29722887
View Article
PubMed/NCBI
Google Scholar

[51] View Article

[52] PubMed/NCBI

[53] Google Scholar

[ref15] 15. Stecher G, Tamura K, Kumar S. Molecular Evolutionary Genetics Analysis (MEGA) for macOS. Mol Biol Evol. 2020; 33:1870–1874. pmid:31904846
View Article
PubMed/NCBI
Google Scholar

[55] View Article

[56] PubMed/NCBI

[57] Google Scholar

[ref16] 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. pmid:26585518
View Article
PubMed/NCBI
Google Scholar

[59] View Article

[60] PubMed/NCBI

[61] Google Scholar

[ref17] 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. pmid:23432962
View Article
PubMed/NCBI
Google Scholar

[63] View Article

[64] PubMed/NCBI

[65] Google Scholar

[ref18] 18. Rodriguez-R LM, Konstantinidis KT. The enveomics collection: a toolbox for specialized analyses of microbial genomes and metagenomes. PeerJ. 2016; 4:e1900v1.
View Article
Google Scholar

[67] View Article

[68] Google Scholar

[ref19] 19. Huson D, Bryant D. Application of phylogenetic networks in evolutionary studies. Mol Biol Evol. 2006; 23:254–267. pmid:16221896
View Article
PubMed/NCBI
Google Scholar

[70] View Article

[71] PubMed/NCBI

[72] Google Scholar

[ref20] 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. https://doi.org/10.1093/bioinformatics/btm404

[ref21] 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. pmid:33349678
View Article
PubMed/NCBI
Google Scholar

[75] View Article

[76] PubMed/NCBI

[77] Google Scholar

[ref22] 22. Delmont TO, Eren AM. Linking pangenomes and metagenomes: the Prochlorococcus metapangenome. PeerJ. 2018; 6:e4320. pmid:29423345
View Article
PubMed/NCBI
Google Scholar

[79] View Article

[80] PubMed/NCBI

[81] Google Scholar

[ref23] 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. pmid:25950237
View Article
PubMed/NCBI
Google Scholar

[83] View Article

[84] PubMed/NCBI

[85] Google Scholar

[ref24] 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. pmid:25118885
View Article
PubMed/NCBI
Google Scholar

[87] View Article

[88] PubMed/NCBI

[89] Google Scholar

[ref25] 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. pmid:24505072
View Article
PubMed/NCBI
Google Scholar

[91] View Article

[92] PubMed/NCBI

[93] Google Scholar

[ref26] 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. pmid:29292687
View Article
PubMed/NCBI
Google Scholar

[95] View Article

[96] PubMed/NCBI

[97] Google Scholar

[ref27] 27. Konstantinidis KT, Tiedje JM. Towards a genome-based taxonomy for prokaryotes. J Bacteriol Res. 2005; 6258–6264. pmid:16159757
View Article
PubMed/NCBI
Google Scholar

[99] View Article

[100] PubMed/NCBI

[101] Google Scholar

[ref28] 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. pmid:30127013
View Article
PubMed/NCBI
Google Scholar

[103] View Article

[104] PubMed/NCBI

[105] Google Scholar

[ref29] 29. Bongrand C, Ruby EG. The impact of Vibrio fischeri strain variation on host colonization. Curr Opin Microbiol. 2019; 50:15–19. pmid:31593868
View Article
PubMed/NCBI
Google Scholar

[107] View Article

[108] PubMed/NCBI

[109] Google Scholar

[ref30] 30. Cole JA, Richardson DJ. Respiration of nitrate and nitrite. EcoSal Plus. 3.2.5, pmid:26443731
View Article
PubMed/NCBI
Google Scholar

[111] View Article

[112] PubMed/NCBI

[113] Google Scholar

[ref31] 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.
View Article
Google Scholar

[115] View Article

[116] Google Scholar

[ref32] 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. pmid:10542156
View Article
PubMed/NCBI
Google Scholar

[118] View Article

[119] PubMed/NCBI

[120] Google Scholar

[ref33] 33. Wang Y, Ruby EG. The roles of NO in microbial symbioses. Cellular Microbiol. 2011; 13:518–526. pmid:21338463
View Article
PubMed/NCBI
Google Scholar

[122] View Article

[123] PubMed/NCBI

[124] Google Scholar

[ref34] 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. pmid:26905381
View Article
PubMed/NCBI
Google Scholar

[126] View Article

[127] PubMed/NCBI

[128] Google Scholar

[ref35] 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. pmid:24405789
View Article
PubMed/NCBI
Google Scholar

[130] View Article

[131] PubMed/NCBI

[132] Google Scholar

[ref36] 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.
View Article
Google Scholar

[134] View Article

[135] Google Scholar

[ref37] 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.
View Article
Google Scholar

[137] View Article

[138] Google Scholar

[ref38] 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. pmid:32663475
View Article
PubMed/NCBI
Google Scholar

[140] View Article

[141] PubMed/NCBI

[142] Google Scholar

Figures

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)

Multilocus sequence analysis (MLSA)

Pan and core genome analyses

In silico chemical taxonomy

Results and discussion

Molecular phylogenetic analysis based on 16S rRNA gene nucleotide sequences

Genomic features and overall genome relatedness indices (OGRIs)

Multilocus sequence analysis (MLSA)

Experimental phenotypic characterization and in silico phenotyping

Pan and core genomes and its ecogenomic interpretation

In silico chemical taxonomy

Conclusions

Description of Neptuniibacter victor sp. nov.

Supporting information

S1 Table. Chemotaxonomic profile of previously described Neptuniibacter.

S1 Fig. Heat map representation of ANI values.

S2 Fig. Heat map representation of AAI values.

S3 Fig. In silico phenotyping of PT1 by Traitar.

S4 Fig. Predicted fatty acid synthetic pathway in Neptuniibacter.

S5 Fig. Gene structure of FAS associated genes.

S6 Fig. Genomic distribution of fab and associated genes.

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

Acknowledgments

References