Vibrio aphrogenes sp. nov., in the Rumoiensis clade isolated from a seaweed

A novel strain Vibrio aphrogenes sp. nov. strain CA-1004T isolated from the surface of seaweed collected on the coast of Mie Prefecture in 1994 [1] was characterized using polyphasic taxonomy including multilocus sequence analysis (MLSA) and a genome based comparison. Both phylogenetic analyses on the basis of 16S rRNA gene sequences and MLSA based on eight protein-coding genes (gapA, gyrB, ftsZ, mreB, pyrH, recA, rpoA, and topA) showed the strain could be placed in the Rumoiensis clade in the genus Vibrio. Sequence similarities of the 16S rRNA gene and the multilocus genes against the Rumoiensis clade members, V. rumoiensis, V. algivorus, V. casei, and V. litoralis, were low enough to propose V. aphrogenes sp. nov. strain CA-1004T as a separate species. The experimental DNA-DNA hybridization data also revealed that the strain CA-1004T was separate from four known Rumoiensis clade species. The G+C content of the V. aphrogenes strain was determined as 42.1% based on the genome sequence. Major traits of the strain were non-motile, halophilic, fermentative, alginolytic, and gas production. A total of 27 traits (motility, growth temperature range, amylase, alginase and lipase productions, and assimilation of 19 carbon compounds) distinguished the strain from the other species in the Rumoiensis clade. The name V. aphrogenes sp. nov. is proposed for this species in the Rumoiensis clade, with CA-1004T as the type strain (JCM 31643T = DSM 103759T).


Introduction
The genus Vibrio, first proposed in 1854, is a large group of bacteria showing Gram negative and with most species requiring salt for growth [2]. Currently 111 Vibrio species have been a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 described (http://www.bacterio.net/) [2]. The genus Vibrio, along with other members of Vibrionaceae, is at the forefront of bacterial taxonomy, having been tested using new methodologies, e.g. amplified fragment length polymorphism (AFLP), multilocus sequence analysis (MLSA), and genome-based sequence comparison [2][3][4][5][6]. Among them, the MLSA has been used as a powerful tool to find "clades" sharing a possible common ancestry among metabolically versatile Vibrionaceae species/strains [3][4][5]. The 8-gene MLSA defines 23 Vibrio and Photobacterium clades and an Enterovibiro-Grimontia-Salini vibrio super clade, which help us to elucidate the dynamic nature of biodiversity and evolutionary history interacting with the Earth's ecosystem [5]. Rapid expansion of genome sequencing methodology in bacterial taxonomy also assists and accelerates the accumulation of our knowledge of vibrio biodiversity and has contributed towards the proposals for new clades within the family Vibrionaceae such as Agarivorans [3] and Swingsii [7].
Vibrio rumoiensis was isolated as a strong catalase producer from the drain pool of a fish processing plant that uses H 2 O 2 as a bleaching and microbial agent [8]. In one of the first uses of MLSA for Vibrionaceae taxonomy, V. rumoiensis was classified as an orphan clade species [4]. Subsequent MLSA showed that V. litoralis, a tidal flats isolate [9], could share a common ancestry with V. rumoiensis which led to the proposal for the Rumoiensis clade [5]. Currently, there are four species known to be a member of the Rumoiensis clade: V. rumoiensis, V. algivorus [10], V. casei [11], and V. litoralis [9]. V. casei, and V. algivorus were isolated from surface of cheeses and the gut of a turban shell, Turbo cornutus, respectively. All species share an assimilation pattern of carbohydrates such as D-mannose, D-galactose, D-fructose, and Dmannitol, nitrate reduction, and, with the exception of V. casei, non-motility [9][10][11]. The ecophysiological coherence of Rumoiensis clade species is still unknown.
A vibrio strain phylogenetically related to the Rumoiensis clade was isolated from the surface of seaweed samples collected from the coast of Mie prefecture, Japan in 1994. This bacterium was originally isolated as a κ-carrageenase producer with a cgk gene [1]. Further phylogenetic, genetic and genomic characterizations in this study revealed the novelty of the strain placing it into the Rumoiensis clade. Importantly, the strain is the first microbe to produce hydrogen from alginate. The gas production is supported by having a hyf-type formate hydrogen lyase gene cluster, the discovery of which is the first in the gas producing species in the Rumoiensis clade. The V. aphrogenes sp. nov. CA-1004 T might hold important clues in elucidating the evolutionary history of species in the Rumoiensis clade and a biotechnological novelty in Vibrionaceae.

Materials and methods
Bacterial strains and phenotypic characterization V. aphrogenes strain CA-1004 T isolated from seaweed surface in 1994 collected at Mie Prefecture in Japan [1] was characterized. For phenotypic characterization, all type strains belonging to the Rumoiensis clade were cultured on ZoBell 2216E agar medium and the phenotypic characteristics were determined according to previously described methods [3].
Phylogenetic analysis based on a 16S rRNA gene A 1400 bp of 16S rRNA gene sequence of the strain CA-1004 T was obtained according to Alsaari et al. [3], using the amplification primers (24F and 1509R) corresponding to positions 25 to 1521 in the Escherichia coli sequence. The other Vibrionaceae sequences used to reconstruct a broad phylogenetic tree shown in S1 Fig were retrieved from the GenBank/DDBJ/EMBL database and analyzed using ClustalX version 2.1 [12] and MEGA version 7.0.16 programs [13]. In the final tree (Fig 1), the 16S rRNA gene sequences of A. fischeri NCIMB 1281 T (X70640), A. logei NCIMB 2252 T (AJ437616), V. algivorus NBRC 111146 T (SA2 T ) (LC060680), V. casei DSM 22364 T (NR_116870), V. litoralis DSM 17657 T (DQ097523), and V. rumoiensis FERM P-14531 T (S-1 T ) (AB013297) were used to reconstruct the tree using the MEGA program with three different methods; neighbor-joining (NJ), maximum parsimony (MP), and maximum likelihood (ML). The robustness of each topology was checked using the NJ method with 500 bootstrap replications. Evolutionary distances of the NJ method were corrected using the Jukes-Cantor method.

Genome sequencing
Draft genome sequences of strain CA-1004 T , V. algivorus NBRC 111146 T , V. casei DSM 22364 T , and V. rumoiensis FERM P-14531 T were obtained using the MiSeq platform. For CA-1004 T only, a paired-end library and an 8 kb mate-pair library were constructed using the Nextera XT DNA Library Preparation Kit and the Nextera Mate Pair Sample Preparation Kit, respectively. Genome sequences of the other strains were obtained from a paired-end library preparing using the Nextera XT DNA Library Preparation Kit for V. aphrogenes and TruSeq PCR-Free kit for V. algivorus, V. casei and V. rumoiensis. The genome sequence was assembled using Platanus [14]. The sequence was deposited in the DDBJ/GenBank/EMBL database under accession numbers described below.

Multilocus sequence analysis (MLSA)
Sequences of eight protein-coding genes (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA, and topA) of CA-1004 T were retrieved from the genome sequences. MLSA was conducted in the same manner as previously described [4][5]. The sequences were aligned using ClustalX 2.1 [12]. The domains used to construct the tree shown in  decomposition analysis (SDA) was performed using SplitsTree version 4.14.3 with a neighbor net drawing and a Jukes-Cantor correction [15][16]. Each aligned set was concatenated and used to reconstruct the network.

DNA-DNA hybridizations
Strains used for DNA-DNA hybridization were CA-1004 T , V. algivorus NBRC 111146 T , V. casei DSM 22364 T , V. litoralis DSM 17657 T , and V. rumoiensis FERM P-14531 T . DNAs of the strains were prepared accordingly to Marmur [17] with minor modifications. DNA-DNA hybridization experiments were performed using the fluorometric direct binding method in microdilution wells described previously [3]. In brief, the DNAs of CA-1004 T were labeled with photobiotin (Vector Laboratories, Inc., Burlingame, CA). After immobilization of unlabeled single stranded DNA of CA-1004 T in microdilution wells (Immuron 200, FIA/LIA plate, black type, Greiner labotechnik, Germany), hybridization was performed under optimal conditions using the CA-1004 T labeled DNA as a probe following pre-hybridization [3]. Detection of the hybridized probe was performed using fluorometry (Infinite 200, Tecan, Switzerland) after binding streptavidin-β-galactosidase to the probe DNA. 4-Methylumbelliferyl-β-D-galactopyranoside (6 x 10 −4 M; Wako, Osaka, Japan) was used for a fluorogenic substrate for β- Vibrio aphrogenes sp. nov., in the Rumoiensis clade isolated from a seaweed galactosidase. DNA-DNA homology was calculated according to the previous report [3] based on an average value measured from three wells.

Genome analysis and in silico DNA-DNA similarity calculation
General genome features including DNA G+C content were determined using the Rapid Annotations Using Subsystems Technology (The RAST server version 4.0) [18]. In silico DDH values from Genome-to-Genome Distance Calculator (GGDC 2) [19][20] and Average Nucleotide Identity (ANI) values of CA-1004 T against V. algivorus NBRC 111146 T , V. casei DSM 22364 T , V. litoralis DSM 17657 T , and V. rumoiensis FERM P-14531 T were estimated using Orthologous Average Nucleotide Identity Tool version 0.93 [21]. Comparison of genes encoding the hyf-type formate hydrogen lyase complex and the flanking region was performed using GenomeTraveler (In Silico Biology, Inc., Yokohama, Japan).

Hydrogen production from alginate
Strain CA-1004 was cultured at 25˚C in a 100 mL marine broth (0.5% (w/v) polypeptone, 0.1% (w/v) yeast extract) containing 100 mM MES (Dojindo, Kumamoto, Japan), supplemented with 1.0% (w/v) sodium alginate. A 3.0% (w/v) mannitol supplemented marine broth was used as a positive control. The pH of the medium was maintained at 6.0 using a pH controller (DT-1023P, ABLE, Tokyo, Japan) equipped with an autoclavable electrode (FermProbe pH electrodes, Broadley-James Corp., Branford, USA) by adding 5 N NaOH or HCl. Biogas was captured in an aluminium bag, and the H 2 gas production was determined using gas chromatography (GC2014 Shimadzu, Kyoto, Japan) with a thermal conductivity detector and a Shin-Carbon ST column (Shinwa Chemical Industries Ltd., Kyoto, Japan).

Results and discussion
The phylogenic analysis based on 16S rRNA gene sequences showed the strain CA-1004 T is a member of the genus Vibrio (S1 Fig): more precisely, the strain was closely related to members of the Rumoiensis clade with a high bootstrap support [4][5] (Fig 1). Sequence similarities of the 16S rRNA gene against those of Rumoiensis clade species, V. algivorus NBRC 111146 T , V. rumoiensis FERM P-14531 T , V. casei DSM 22364 T , and V. litoralis DSM 17657 T were 98.4%, 98.0%, 97.9%, and 96.8%, respectively. These levels of similarity are below or in the proposed threshold range for the species boundary, 98.2-99.0% [22,23,24]. To further confirm the genetic coherence, DNA-DNA similarity of CA-1004 T against Rumoiensis clade species was experimentally measured. Using CA-1004 T as a labelled strain, DDH values against V. algivorus NBRC 111146 T , V. casei DSM 22364 T , V. litoralis DSM 17657 T , and V. rumoiensis FERM P-14531 T were 15.4%, 12.0%, 4.9%, and 5.6%, respectively. These DDH values were sufficiently below the species boundary (<70%) to propose CA-1004 T as a new species in the Rumoiensis clade. MLSA using eight protein-coding genes also showed the clear separation of the Rumoiensis clade containing the CA-1004 T from the other clades of Vibrionaceae species, which suggests a common ancestry of the CA-1004 T and the other Rumioensis clade species (Fig 2, S2 Fig).
On the basis of concatenated sequences of eight MLSA protein-coding genes, CA-1004 T is likely to share a common ancestry with members of the Rumoiensis clade. This was confirmed by comparative analysis of phenotypic and biochemical features of CA100-4 T with other members of the Rumoiensis clade (Table 1), showing some degree of phenotypic coherence between the different species. They grow at temperatures between 4 and 30˚C, require salt for growth, and tested positive for nitrate reduction, oxidase, catalase, DNase, and alginase production. They were negative for growth on TCBS agar, lysine and ornithine decarboxylation, acetoin production, and indole production. On the other hand, CA-1004 T was distinguished from the close neighbors by several traits, such as showing positive results for gas production from Dglucose and arginine dihydrolase. Apparent κ-carrageenase activity reported by Araki et al. [1] was detected in the type strain proposed, but no any κ-carrageenase activities or cgk genes Vibrio aphrogenes sp. nov., in the Rumoiensis clade isolated from a seaweed were retained in the draft genome sequence. The genes may have been lost during the long term serial transfers. The five species belonged into the Rumoiensis clade can grow wide range of NaCl concentration (Table 1).
Interestingly, the strain CA-1004 T possessed an entire gene set responsible for a hyf-type formate hydrogen lyase complex [27] (Fig 4A). The gene cluster consisted of genes for a major All species were Gram negative, fermentative, require salt for growth, and oxidase-and catalase-positive. All species were positive for growth on 4, 15, 25, 30˚C, growth in 1, 3, 6, 8, 10% NaCl broth, DNase, nitrate reduction, and utilization of D-mannose, D-galactose, fumarate, D-mannitol, glycerol, acetate, pyruvate, Lproline, L-alanine, L-asparagine, and L-serine. All species were negative for pigmentation, growth on TCBS, acetoin production, indole production, agarase, gelatinese, κ-carrageenase, lysine and ornithine decarboxylase, bioluminescence, and utilization of N-acetylglucosamine, succinate, citrate, aconitate, γaminobutyrate, L-tyrosine, D-sorbitol, DL-malate, amygdalin, α-ketoglutarate, trehalose, δ-aminovarate, Lglutamate, putrescine, D-galacturonate, DL-lactate, L-citrulline, and glycine. https://doi.org/10.1371/journal.pone.0180053.t001 Vibrio aphrogenes sp. nov., in the Rumoiensis clade isolated from a seaweed part of FHL (a hydrogenase complex and a formate dehydrogenase), and the flhA activator gene, which corresponds to the FHL-Hyp gene cluster of V. tritonius AM2 T [27]. The presence of the gene cluster supports the gas production phenotype of the strain. In addition to the gene cluster, a possible nickel transporter gene, hupE, was located between the formate dehydrogenase gene and the hyp gene cluster (Fig 4A). The V. aphogenes-type of FHL gene cluster Vibrio aphrogenes sp. nov., in the Rumoiensis clade isolated from a seaweed involving the hupE gene is also found in Gazogenes clade species including V. gazogenes ( Fig  4A) but the hydrogen productions by these strains are rather low (unpublished data). The biochemistry and molecular biology of HupE in the V. aphrogenes CA-1004 T have not been investigated yet, but the function of the R. leguminosarum HupE is recently identified as an energyindependent and specific diffusion facilitator for nickel transport for hydrogenase synthesis, on the basis of the kinetics using inhibitors and uncouplers such azide, arsenate, CCCP, and DCCD in the Rhizobium hydrogen uptake system [28][29]. Mutant assays also revealed good correlation between the nickel transport and the hydrogenase activity in R. leguminosarum [29]. The hydrogenase of V. aphrogenes is predicted as a [NiFe] hydrogenase on the basis of the primary structure comparison possessing CxxC motif required for [NiFe] center construction [27], the nickel transport via the HupE could have a strong link to the hydrogenase activity. Further genome comparison with other members of Rumoiensis clade revealed no any FHL-fdhF-hyp gene cluster. Other members of Rumoiensis possessed regions containing serine/threonine protein kinase, RIO1 family protein gene, replacing 66-kb genome region with FHL-fdhF-hyp gene cluster in V. aphrogenes ( Fig 4B). As the gas production is an atypical phenotype not only in the genus Vibrio but also in the family Vibrionaceae [2,[4][5]30], this might give important clues in revealing the evolutionary history of hyf-type gene cluster present in vibrios.
More interestingly, we found a direct hydrogen production (2.9 mL H 2 gas at 25˚C at 48 hours) by the V. aphrogenes strain CA-1004 T from alginate, which is major polysaccharide in brown seaweed. As alginate is known as one of the most oxidized polysaccharides, reduced fermentation products such as ethanol and lactate are unlikely to be produced from such substrate during the fermentation of bacteria due to the redox imbalance [31][32]. Since H 2 is also known to be a reduced fermentation gaseous product, no bacteria possessing direct alginate-H 2 conversion metabolisms have been reported until now. The new findings of the V. aphrogenes sp. nov. could illuminate the future metabolic pathway designs in H 2 production even when using redox imbalanced substrates. We need further characterization to show how direct H 2 production from alginate is controlled genetically and/or biochemically in this unique Vibrio species for future applications of V. aphorogenes.
In conclusion, polyphasic taxonomy with a genome-based strategy indicated V. aphrogenes as a new species in the genus Vibrio. Both 16S rRNA gene sequences phylogeny and MLSA based on eight protein-coding gene sequences placed the stain CA-1004 T into the Rumoiensis clade. Comparison of phenotypic features also places V. aphrogenes CA-1004 T in the genus Vibrio, while supporting its novelty ( Table 1). The name V. aphrogenes is proposed to show its gas-producing features. Unfortunately we have only one strain of V. aphrogenes today, further ecological study is necessary for understanding the biodiversity and ecophysiological roles of the V. aphrogenes strains.
Description of Vibrio aphrogenes sp. nov.