Zobellia barbeyronii sp. nov., a New Member of the Family Flavobacteriaceae, Isolated from Seaweed, and Emended Description of the Species Z. amurskyensis, Z. laminariae, Z. russellii and Z. uliginosa

Six Gram-stain-negative, aerobic, rod-shaped, and motile by gliding bacterial strains were isolated from Pacific green and red algae. Phylogenetic analysis based on 16S rRNA gene sequences placed the novel strains into the genus Zobellia as a distinct evolutionary lineage close to Zobellia nedashkovskayae Asnod2-B07-BT and Zobellia laminariae KMM 3676T sharing the highest similarity of 99.7% and 99.5%, respectively. The average nucleotide identity and the average amino acid identity values between strains 36-CHABK-3-33T and Z. nedashkovskayae Asnod2-B07-BT and Z. laminariae KMM 3676T were 89.7%/92.9% and 94.2%/95.8%, respectively. The digital DNA–DNA hybridization values based on the draft genomes between strains 36-CHABK-3-33T and Z. nedashovskayae Asnod2-B07-BT and Z. laminariae KMM 3676T were 39.5 ± 2.5% and 59.6 ± 2.7%, respectively. Multilocus sequence analysis based on house-keeping genes (dnaK, gyrB, pyrH, recA and topA) assigned the alga-associated isolates to the same species, which clustered separately from the recognized species of the genus Zobellia. The strains under study grew at 4–32 °C and with 0.5–8% NaCl and decomposed aesculin, gelatin, DNA, and Tweens 20 and 80, and weakly agar. The DNA G+C content was 36.7% calculated from genome sequence analysis for the strain 36-CHABK-3-33T. The predominant fatty acids of strain 36-CHABK-3-33T (> 5% of the total fatty acids) were iso-C17:0 3-OH, summed feature 3 (comprising C16:1 ω7c and/or iso-C15:0 2-OH fatty acids), iso-C15:0, iso-C15:1 G, and C15:0. The major polar lipids were phosphatidylethanolamine, three unidentified lipids, and two unidentified aminolipids. The only detected respiratory quinone was MK-6. The significant molecular distinctiveness between the novel isolates and their nearest neighbor was strongly supported by differences in physiological and biochemical tests. Therefore, the six novel strains represent a novel species of the genus Zobellia, for which the name Zobellia barbeyronii sp. nov. is proposed. The type strain is 36-CHABK-3-33T (= KACC 21790T = KMM 6746T).


Introduction
The genus Zobellia was first described by Barbeyron et al. [1] with Zobellia galactanivorans as the type species. At the time of writing, the genus Zobellia comprises seven species with validly published names (as listed at [2]) that were recovered from different marine environments and have a DNA G+C content of 36.7-42.8 mol%. Species in the genus Zobellia are Gram negative, aerobic, heterotrophic, rod-shaped, non-spore-forming, and gliding bacteria that form orange or yellow colonies and produce flexirubin-type pigments. Most species possess agarolytic activity [1,3,4]. The type strain of the type species of the genus, Z. galactanivorans, was isolated from the red alga Delesseria sanguinea, collected in the English Channel near Roscoff (Brittany, France) [1]. Strains of species Z. laminariae and Z. russellii were also isolated from seaweeds: the brown alga Saccharina (formerly Laminaria) japonica and the green alga Acrosiphonia sonderi, respectively, the widespread algae inhabiting the Sea of Japan, the Pacific Ocean [3]. Strains of the recently described species, Z. roscoffensis and Z. nedashkovskayae, were members of the epiphytic communities of the brown alga Ascophyllum nodosum collected in Roscoff (Brittany, France) too [4]. Unlike the above, species Z. amurskyensis was recovered from a seawater sample, collected from Amursky Bay of the Sea of Japan [3]. The species Z. uliginosa, isolated from the surface sediment and originally named as Flavobacterium uliginosum [5], was subsequently placed in the genus Cytophaga as Cytophaga uliginosa [6] and to the genus Cellulophaga as Cellulophaga uliginosa [7]. At present, it is classified in the genus Zobellia because of the close phylogenetic relatedness to members of the genus along with the DNA G+C content, maximum growth temperature, and the presence of flexirubin type pigments [1]. The studies of the genomes of bacteria belonging to the genus Zobellia have confirmed the results of phenotypic tests obtained previously, and significantly expanded our knowledge of the ability of bacteria of this taxonomic group to utilize polysaccharides of marine algae [4,[8][9][10]. Moreover, the type strain of the species Z. galactanivorans has been proposed as a model organism for the study of bacteria-algae interactions [9].
In the present work, we report the phenotypic, genomic, and phylogenetic characterization of six Gram-negative, aerobic, dark-orange, motile by gliding, and agarolytic bacterial strains, which were recovered from a green alga Ulva sp. and an unidentified red alga during a survey of the biodiversity of microbial communities of marine organisms living in isolation in Kraternaya Bay near volcano Ushishir (Kuril Islands, Russia). The detail taxonomic analysis based on a polyphasic approach indicated that these alga-associated isolates represent a novel species of the genus Zobellia.

Bacterial Isolation and Cultivation
Strains 36-CHABK-3-33 T , 36-CHABK-3-51, 36-CHABK-3-57, and 36-CHABK-3-61 were isolated from the green alga Ulva sp., and strains 36-RHABK-5-24 and 36-RHABK-5-54 were isolated from an unidentified red alga (raised from a depth of 53 m). All of them were collected in the Kraternaya Bay, Yanchich Island, Kuril Islands, the Sea of Okhotsk (47.510239 N; 152.822071 E) during 36th cruise of R/V "Academic Oparin" in August 2008. The samples of algal fronds (5 g) were washed twice with sterile seawater to remove loosely attached bacteria and homogenized in 10 mL sterile seawater in a glass homogenizer and 0.1 mL homogenate was spread onto marine agar 2216 (MA, BD, Difco, Sparks, MD, USA) plates using a dilution plating method. Each novel isolate was obtained from a single colony after incubation of the plate at 28 • C for 7 days. After primary isolation and purification, the strains were cultivated at 25-28 • C on the same medium and stored at -80 • C in marine broth (Difco) supplemented with 20% (v/v) glycerol.

16S rRNA Gene Sequencing and Phylogenetic Analysis
DNAs were extracted from bacterial cultures using the NucleoSpin Microbial DNA Mini kit (Macherey-Nagel, Düren, Germany), following the manufacturer's instructions. The 16S rRNA genes were amplified with the 27F (5 -AGAGTTTGATCMTGGCTCAG-3 ) and 1492R (5 -TACGGTTACCTTGTTACGACTT-3 ) primers [11] and were sequenced on an ABI Prism 3130 xL DNA analyzer (Applied Biosystems, Hitachi, Japan) using the Big Dye Terminator reagent kit, version 3.1. The gene sequences of the strains were deposited in GenBank/EMBL/DDBJ under the accession numbers from MZ890274 to Diversity 2021, 13, 520 3 of 13 MZ890278. The 16S rRNA gene sequence analysis for identification of the strains was performed using the EzBioCloud [12]. The 16S rRNA gene sequences were aligned by Clustal W [13]. Phylogenetic analysis was carried out using the maximum-likelihood (ML), neighbor-joining (NJ), and maximum-parsimony (MP) algorithms implemented in the MEGA7 software [14]. The genetic distances were calculated according to the Kimura twoparameter model [15]. Bootstrap values were calculated from 500-1000 alternative trees.

Multilocus Sequence Analysis (MLSA)
The multilocus sequence analysis (MLSA) was conducted using concatenated sequences of five housekeeping genes, namely dnaK (Chaperone protein DnaK), gyrB (DNA gyrase, B subunit), pyrH (Uridylate kinase), recA (Recombinase A), and topA (DNA topoisomerase I). The MLSA primers were designed based on genome sequence of the 36-CHABK-3-33 T using the Vector NTI software package version 11.0 (Invitrogen, Carlsbad, CA, USA) (Table S1). Primer sequences were examined further to confirm that no secondary structures were likely to form. The sequences of strain 36-CHABK-3-33 T and other members of the genus Zobellia were retrieved from their whole genome sequences. The partial sequences of the remaining five isolates were obtained by amplification and sequencing of the corresponding genes. The gene sequences of five strains were deposited in GenBank/EMBL/DDBJ under the accession numbers from MZ911872 to MZ911896. The sequences obtained were then concatenated, aligned, and used to reconstruct ML, MP, and NJ phylogenetic trees in the MEGA7 software [14]. The genetic variability across the five genes of the MLSA analyses was estimated using the Kimura 2-parameter model with 1st+2nd+3rd+Noncoding codon positions. Split decomposition analysis was performed for the concatenated genes using SplitsTree version 4.14.3 with a neighbor net drawing and a Jukes-Cantor correction [16].

Genome Features of Strain 36-CHABK-3-33 T and Phylogenomic Reconstruction
The genomic DNA was obtained from the strain 36-CHABK-3-33 T cells as described above in the Section 2.2 using the NucleoSpin Microbial DNA Mini kit (Macherey-Nagel, Düren, Germany). Whole-genome shotgun sequencing of the strain 36-CHABK-3-33 T was carried out at an Illumina MiSeq platform using Nextera DNA Flex kits (Illumina, San Diego, CA, USA) and a 150-bp paired-end kit (Illumina, San Diego, CA, USA). The sequence quality was assessed via FastQC version 0.11.8 [17] and reads were trimmed using Trimmomatic version 0.38 [18]. Filtered reads were assembled de novo with SPAdes version 3.13.1 [19] and assembly metrics were calculated with QUAST version 5.0.2 [20]. The genome completeness was further evaluated by checkM version 1.1.3 based on the taxonomic-specific workflow (family Flavobacteriaceae) [21]. The draft genome of strain 36-CHABK-3-33 T was annotated using NCBI Prokaryotic Genome Annotation Pipeline (PGAP) [22] and its features are summarized in Table 1. The genome of strain 36-CHABK-3-33 T was deposited in GenBank/EMBL/DDBJ under the accession number JACATN000000000.1.
The phylogenetic analysis was performed with PhyloPhlAn 3.0 using 400 conserved protein sequences [23]. The Average Nucleotide Identity (ANI) and Amino Acid Identity (AAI) values were calculated with the online server ANI/AAI-Matrix [24]. Values of in silico DNA-DNA hybridization (dDDH) of the strain 36-CHABK-3-33 T and its closest relatives were measured at TYGS platform [25]. To identify carbohydrate-active enzymes (CAZymes) in the Zobellia genomes, we used the dbCAN2 meta server (http://cys.bios. niu.edu/dbCAN2, accessed on 18 December 2020) [26]. Predictions by at least one of the three algorithms integrated within the server (DIAMOND, HMMER, and Hotpep) were considered sufficient for CAZy family assignments.
For determination of whole-cell fatty acid and polar lipid profiles, strains 36-CHABK-3-33 T , Z. galactanivorans CIP 106680 T , Z. amurskyensis KMM 3526 T , Z. laminariae KMM 3676 T , Z. russellii KMM 3677 T , and Z. uliginosa DSM 2061 T were grown for 48 h on MA. Cellular fatty acid methyl esters were prepared according to the standard protocol of the Microbial Identification System (MIDI, version 3.5) [30] and analysed using a GC-17A chromatograph (Shimadzu, Kyoto, Japan) equipped with a fused silica capillary column (30 m × 0.25 mm) coated with Supercowax-10 and SPB-5 phases (Supelco, Bellefonte, PA, USA) at 210 • C. FAMES were identified using equivalent chain length measurements and by comparing of retention times those of authentic standards. FAMEs were also analysed by GC-MS (Shimadzu QP5050A) equipped with an MDN-5S capillary column (30 m × 0.25 mm) at temperature of the injector and detector of 250 • C. Polar lipids were determined by TLC as described by Minnikin et al. [31].

Phylogenetic Analysis
Nearly full-length 16S rRNA gene sequences (1384 bp) of the six isolates were determined and aligned with corresponding sequences of members of the genus Zobellia, which were retrieved from genomic sequences. The phylogenetic trees based on 16S rRNA gene sequences reconstructed with NJ, ML, and MP algorithms showed highly similar branch topology. According to the ML tree (Figure 1), the six isolates clustered together, and were most closely related to the Z. nedashkovskayae (99.7% of sequence similarity), followed by Z. laminariae (99.5%), and Z. amurskyensis (98.8%).

Multilocus Sequence Analysis of the Alga-Associated Isolates
The 16S rRNA sequences provided insufficient phylogenetic resolution of the six isolates at the strain level. To elucidate phylogenetic relationships among the six isolates without whole genome sequencing, we developed an MLSA based on the five housekeeping genes: dnaK, gyrB, pyrH, recA, and topA. In addition, we performed a concatenated sequence analysis because it can more accurately predict intraspecific relationships. Based on the ML, MP, and NJ phylogenies ( Figure S1), and split tree decomposition analysis (Figure 2), the MLSA placed all isolates in a separate branch within the clade with Z. laminariae and Z. nedashkovskayae. The resulting tree also confirmed the branch topology of the 16S rRNA gene tree and the genetic variability of the six alga-associated isolates. The MLSA distances between the isolates were 0.000-0.014 (average value was 0.010 ± 0.002), and between the isolates and two type strains Z. nedashkovskayae and Z. laminariae were 0.053 ± 0.005 and 0.055 ± 0.005, respectively (Table S2). The phylogenetic analysis revealed that the six strains belong to the genus Zobellia and cluster with Z. nedashkovskayae Asnod2-B07-B T but form a separate branch supported by high bootstrap values of 99%. It indicates that the novel strains could represent a new species of the genus Zobellia.

Multilocus Sequence Analysis of the Alga-Associated Isolates
The 16S rRNA sequences provided insufficient phylogenetic resolution of the six isolates at the strain level. To elucidate phylogenetic relationships among the six isolates without whole genome sequencing, we developed an MLSA based on the five housekeeping genes: dnaK, gyrB, pyrH, recA, and topA. In addition, we performed a concatenated sequence analysis because it can more accurately predict intraspecific relationships. Based on the ML, MP, and NJ phylogenies ( Figure S1), and split tree decomposition analysis (Figure 2), the MLSA placed all isolates in a separate branch within the clade with Z. laminariae and Z. nedashkovskayae. The resulting tree also confirmed the branch topology of the 16S rRNA gene tree and the genetic variability of the six alga-associated isolates. The MLSA distances between the isolates were 0.000-0.014 (average value was 0.010 ± 0.002), and between the isolates and two type strains Z. nedashkovskayae and Z. laminariae were 0.053 ± 0.005 and 0.055 ± 0.005, respectively (Table S2).

Multilocus Sequence Analysis of the Alga-Associated Isolates
The 16S rRNA sequences provided insufficient phylogenetic resolution of the six isolates at the strain level. To elucidate phylogenetic relationships among the six isolates without whole genome sequencing, we developed an MLSA based on the five housekeeping genes: dnaK, gyrB, pyrH, recA, and topA. In addition, we performed a concatenated sequence analysis because it can more accurately predict intraspecific relationships. Based on the ML, MP, and NJ phylogenies ( Figure S1), and split tree decomposition analysis (Figure 2), the MLSA placed all isolates in a separate branch within the clade with Z. laminariae and Z. nedashkovskayae. The resulting tree also confirmed the branch topology of the 16S rRNA gene tree and the genetic variability of the six alga-associated isolates. The MLSA distances between the isolates were 0.000-0.014 (average value was 0.010 ± 0.002), and between the isolates and two type strains Z. nedashkovskayae and Z. laminariae were 0.053 ± 0.005 and 0.055 ± 0.005, respectively (Table S2).

Genomic Characteristics and Phylogenomic Analysis
The 16S rRNA gene analysis showed close relationship of the six bacterial isolates to Z. nedashkovskayae Asnod2-B07-B T , sharing about 99.7% of sequence similarity. Therefore, the whole genome sequence of strain 36-CHABK-3-33 T was determined using Illumina MiSeq platform. The draft genome was de novo assembled into 37 contigs, with a N50 value of 927,759 bp, and a L50 value of three ( Table 1). The genome size was estimated at 4,977,540 bp in length with a coverage of 24 ×. The G+C content was 36.7 mol%. The genome size and G+C content were within the range of values typical for the genus Zobellia, which ranged from 36.7 to 42.8 mol%, and from 4.92 to 5.52 Mb, respectively [10].
The characteristics of strain 36-CHABK-3-33 T draft genome were compared to those of publicly available genomes of other Zobellia species. The genome completeness was 100% which is comparable with the estimated completeness of other Zobellia genomes, suggesting high assembly quality with of 0-1.07% of contamination among all the genomes ( Table 1).
The genomic tree constructed with PhyloPhlAn3.0 [23] using concatenated sequences of 400 conserved proteins clarified the phylogenetic position of the novel species Z. barbeyronii, which is closer to Z. laminariae than to Z. nedashkovskayae (Figure 3).

Morphological, Physiological and Biochemical Characteristics
The detailed phenotypic characteristics of the new strains are given in Table 2 and in the species description. The alga-associated isolates possessed several common properties with validly published species of the genus Zobellia, including the requirement of sea salts or seawater for growth, hydrolysis of aesculin, agar, gelatin, and tyrosine and production of flexirubin-type pigments (Table 2). However, they could be differentiated from their closest relative, Z. laminariae, by their ability to degrade DNA, Tweens 20 and 80, and to produce acid from D-xylose ( Table 2). The maximum temperature and minimum salinity for growth, along with production of gelatinase and α-chymotrypsin clearly separated the strains studied from another nearest neighbor, Z. nedashkovskayae (Table 2). Several phenotypic traits presented in Table 2 can be helpful for discrimination of the new strains from the type species of the genus Zobellia.  (1) alga-associated isolates (n = 6); (2) Zobellia nedashkovskayae Asnod2-B07-B T and Asnod3-E08-A; (3) Zobellia laminariae KMM 3676 T ; (4) Zobellia galactanivorans CIP 106680 T . All strains were positive for the following tests: gliding motility, catalase, oxidase, alkaline phosphatase, leucine arylamidase, valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, β-galactosidase, β-glucosidase and N-acetyl-β-glucosamidase activities, nitrate reduction, hydrolysis of aesculin and tyrosine, production of flexirubin-type pigments, production of brown pigment on tyrosine, utilization of L-arabinose, D-glucose, maltose, D-mannose, and mannitol, susceptibility to carbenicillin, lincomycin and rifampicin, resistance to benzylpenicillin, kanamycin, neomycin, oxacillin and polymyxin B. All strains were negative for: acetoin, indole and H 2 S production, lipase (C14) and β-glucuronidase activities. Data were obtained from this study unless indicated. v "variable reaction"; *, data from Barbeyron et al. [4].

Conclusions
Phylogenetic analyses based on the 16S rRNA gene sequences of members of the genus Zobellia indicated that the novel strains form a distinct lineage within the genus (Figure 1). The above-mentioned molecular distinctiveness taken together with differences in physiological and biochemical characteristics, and in polar lipid and fatty acid compositions strongly suggest the separate taxonomic status of the novel strains. On the basis of the combined phylogenetic, genotypic, chemotaxonomic, and phenotypic data presented here, we propose that the six alga-associated isolates should be classified as the representatives of a novel species of the genus Zobellia. Based on new data obtained in this study, emended descriptions of the species Z. amurskyensis, Z. laminariae, Z. russellii, and Z. uliginosa are also provided.

Conclusions
Phylogenetic analyses based on the 16S rRNA gene sequences of members of the genus Zobellia indicated that the novel strains form a distinct lineage within the genus (Figure 1). The above-mentioned molecular distinctiveness taken together with differences in physiological and biochemical characteristics, and in polar lipid and fatty acid compositions strongly suggest the separate taxonomic status of the novel strains. On the basis of the combined phylogenetic, genotypic, chemotaxonomic, and phenotypic data presented here, we propose that the six alga-associated isolates should be classified as the representatives of a novel species of the genus Zobellia. Based on new data obtained in this study, emended descriptions of the species Z. amurskyensis, Z. laminariae, Z. russellii, and Z. uliginosa are also provided.
Emended description of the species Zobellia amurskyensis-the description is as given by Nedashkovskaya et al. [3] with the following emendation. The polar lipid profile consists of phosphatidylethanolamine, two unidentified aminolipids, and two unidentified lipids.
Emended description of the species Zobellia laminariae-the description is as given by Nedashkovskaya et al. [3] with the following emendation. The polar lipid profile consists of phosphatidylethanolamine, two unidentified aminolipids, and three unidentified lipids.
Emended description of the species Zobellia russellii-the description is as given by Nedashkovskaya et al. [3] with the following emendation. The polar lipid profile consists of phosphatidylethanolamine, three unidentified aminolipids, and two unidentified lipids.
Emended description of the species Zobellia uliginosa-the description is as given by description was previously given by Reichenbach for [Cytophaga] uliginosa [6] and by Barbeyron et al. [1] with the following emendation. The polar lipid profile consists of phosphatidylethanolamine, two unidentified aminolipids, and two unidentified lipids.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/d13110520/s1, Table S1: MLSA primers designed based on genome sequence of the 36-CHABK-3-33T used for estimation of intraspecies genetic variability, Figure S1: Maximum-likelihood (ML) phylogenetic tree based on concatenated partial dnaK-gyrB-pyrH-recA-topA (2570 bp) gene sequences, showing the phylogenetic position of six alga-associated isolates and members of the genus Zobellia, Table S2: MLSA distances values for the selected strains in this study.