Winogradskyella luteola sp.nov., Erythrobacter ani sp. nov., and Erythrobacter crassostrea sp.nov., isolated from the hemolymph of the Pacific Oyster Crassostrea gigas

Three new bacterial strains, WHY3T, WH131T, and WH158T, were isolated and described from the hemolymph of the Pacific oyster Crassostrea gigas utilizing polyphasic taxonomic techniques. The 16S rRNA gene sequence analysis revealed that strain WHY3T was a member of the genus Winogradskyella, whereas strains WHI31T and WH158T were members of the genus Erythrobacter. According to the polygenomic study the three strains formed individual lineages with strong bootstrap support. The comparison of dDDH-and ANI values, percentage of conserved proteins (POCP), and average amino acid identity (AAl) between the three strains and their relatives established that the three strains represented two separate genera. Menaquinone-6 was reported as the major respiratory quinone in strain WHY3T and Ubiquinone-10 for strains WH131T and WH158T, respectively. The major cellular fatty acids for strain WHY3T were C15:0, anteiso-C15:1 ω7c, iso-C15:0, C16:1ω7c. The major cellular fatty acids for strains WH131T and WH158T were C14:02-OH and t18:1ω12 for WH131T and C17:0, and C18:1ω7c for strain WH158T. Positive Sudan Black B staining Indicated the presence of polyhydroxyalkanoic acid granules for strains WH131T and WH158T but not for strain WHY3T. The DNA G + C contents of strains WHY3T, WH131T and WH158T were 34.4, 59.7 and 56.6%, respectively. Gene clusters predicted some important genes involved in the bioremediation process. Due to the accomplishment of polyphasic taxonomy, we propose three novel species Winogradskyella luteola sp.nov. (type strain WHY3T = DSM 111804T = NCCB 100833T), Erythrobacter ani sp.nov. (WH131T = DSM 112099T = NCCB 100824T) and Erythrobacter crassostrea sp.nov. (WH158T = DSM 112102T = NCCB 100877T). Supplementary Information The online version contains supplementary material available at 10.1007/s00203-022-03099-y.


Introduction
Pacific oyster Crassostrea gigas is the most globally diverse in various environments. This kind of Oyster currently produces more than any other aqua product in the world. The Pacific oyster has the ability to significantly modify its habitat, benefiting and harming native species and ecosystems alike. It is an important invasive aquaculture species that has the potential to outcompete native species and as a habitat-forming species to function as a stepping stone for other non-native species (e.g., the brown algae Sargassum muticum in the North Sea).
Pacific oysters are ecosystem engineers, having a significant physical influence on their habitats (Dumbauld et al. 2009). C. gigas forms thick mats known as oyster reefs, and when they reach a certain density, they trigger physical changes in the surrounding environment. Oysters are well-known for their capacity to transform soft substrates such as mud and silt into hard substrates. Numerous studies of oyster beds in various places have shown larger concentrations of benthic invertebrates, such as crabs, bivalves, and worms, living on the oyster beds' hard substrate compared to the surrounding soft substrate (Dumbauld et al. 2009). They also provide a hard substrate for various macroalgae to grow on. Besides altering the benthic environment, oysters are filter feeders, consuming suspended plankton and organic debris. It has been a source of great interest for the study of associated marine bacteria due to their accumulation in various tissues such as the gills, gastrointestinal tissues, and mantle (Li et al. 2017), as well as in the circulatory fluid system, the hemolymph, which has been targeted as the most promising component to study oyster microbiota due to its role in the immune system (Li et al. 2017). Nedashkovskaya et al. identified the genus Winogradskyella, a member of the family Flavobacteriaceae in the phylum Bacteroidetes (He et al. 2019). The genus Winogradskyella has 44 species with validly published names. Following several revisions of the genus description, the genus is now defined as catalase-positive, strictly aerobic or facultatively anaerobic, motile by gliding, yellowish to orange-colored, rod-or cocci-shaped bacteria that contain phosphatidylethanolamine as a major polar lipid and menaquinone-6 (MK-6) as a major respiratory quinone (Song et al. 2018).
Shiba and Simidu erected the bacterium genus Erythrobacter under the family Erythrobacteraceae (Lee et al. 2005) with the identification of a single type species of Erythrobacter longus (Shiba et al. 1982). Erythrobacter is currently comprised of 12 species with validly published names (Xu et al. 2020). They are abundant in marine habitats and have been isolated and identified largely from tidal fat deposits, marine cyanobacterial mats, and marine plants (Shiba and Simidu 1982;Park et al. 2020). Significant features of Erythrobacter species include negative Gram staining, aerobic or facultatively anaerobic chemoorganotrophic metabolisms, and the synthesis of ubiquinone-10 as their predominant isoprenoid quinone (Tonon et al. 2014).
The bioremediation procedure indicates the use of microorganisms' metabolic ability to clean up polluted environments. It refers to microorganisms' metabolic capacity to mineralize or change organic pollutants into less hazardous chemicals that may be incorporated into natural biogeochemical cycles. Marine bacteria are organisms that are naturally exposed to harsh environments. Therefore, marine bacteria with bioremediation capacity might be great candidates for the biological treatment of harsh contaminated ecosystems. For instance, copper becomes toxic in higher concentrations (for example, > 0.08 µM Cu), impairing the metabolic activities of marine organisms. Bioremediation is a cost-effective method of treating polluted environments. Copper sorption by highly efficient bacteria may be employed to remove copper from polluted locations (Leal et al. 2018).
In this study, we isolated and performed the polyphasic taxonomy of three novel species belonging to the Erythrobacter, and Winogradskyella genus (Strains WHY3 T , WH131 T and WH158 T ) isolated from the hemolymph of the Pacific oyster Crassostrea gigas and, based on genome data, predicted gene clusters for bioremediation and other important processes like polyhydroxyalkanoic acid.

Isolation
The samples were collected of wild oysters from the Wadden Sea near Wilhelmshaven, Germany (Latitude: 53.5131, Longitude: 08.14714) in December 2019, both valves were cleaned externally with a brush and sterile water to eliminate any dirt or debris that may contaminate the extraction process. The adductor muscle was entirely excised with a scalpel blade, and the remainder of the tissues were pooled together. The adductor muscle can be used to capture the hemolymph contained within (King et al. 2019). Serial dilutions of samples were performed (1:10) to a 10 -5 dilution using 100 µL of sample and 900 µL sterile water in 1.5 mL Eppendorf tubes. Artificial saltwater medium (ASW) supplemented with vitamin, and antifungal agent (ATI Coral Ocean salt (39 g/L), agar (15 g/L), nicotinic acid (20 mg/L), thiamine (vitamin B1, 10 mg/L), biotin (vitamin B7, 2 mg/L), 4-aminobenzoic acid (10 mg/L), pantothenic acid (5 mg/L), pyridoxamine (vitamin B6, 50 mg/L), cyanocobalamin (vitamin B12, 20 mg/L), and cycloheximide (100 mg/L), pH 7.3) was used to perform preliminary isolation. The incubation was performed for 6 days at 30 ℃.
The orange and yellow colonies (strains WHY3 T , WH131 T , and WH158 T ) were selected and transferred to Bacto marine agar (MA, Difco 2216), where they were purified successively by streaking over the same medium. The strains were held at − 80 ℃ for long-term preservation.

16S rRNA gene analysis
The Invisorb Spin Plant Mini Kit was used to extract genomic DNA in accordance with the manufacturer's instructions (Stratec Molecular, Germany).PCR amplification of the 16S rRNA gene was applied with the primer F27 (5′-AGA GTT TGATCMTGG CTC AG-3′) and 1492R (5′-TAC GGY TAC CTT GTT ACG ACTT-3′) (Chaiya et al. 2019). The 16S rRNA gene was sequenced employing an Applied Biosystems 3730XL automated sequencer (ABI). BioEdit software was used to modify and assemble the sequence (version 7.0.5.3) (Hall 1999). The 16S rRNA gene sequence of strains WHY3 T (1343 bp), WH131 T (1443 bp), and WH158 T (1486 bp) were almost completely sequenced and submitted to GenBank under the accession number MW888983, MW888981, and MW888982, respectively. The phylogenetically closest strains of WHY3 T , WH131 T , and WH158 T were determined based on 16S rRNA gene sequence similarity using the EZBioCloud system (https:// www. ezbio cloud. net/) . Phylogenetic analysis of the 16S rRNA gene of strains WHY3 T was inferred using the GGDC web server for closely similar type strains (http:// ggdc. dsmz. de/) (Meier-Kolthoff et al. 2013). The sequence was analyzed using a single-gene adaptation of the DSMZ phylogenomics program (Meier-Kolthoff et al. 2014). Multiple sequence alignment was performed using MUSCLE (Edgar 2004). Randomized Axelerated Maximum Likelihood (RAxML) (Stamatakis 2014) and TNT (Tree analysis using New Technology) (Goloboff et al. 2008) programs were applied to estimate Maximum likelihood (ML) and Maximum parsimony (MP) trees, respectively. We employed rapid bootstrapping using the autoMRE (extended majority rule) bootstrapping parameters (Pattengale et al. 2010). 1000 bootstrapping replicates, tree bisection, reconnection branch switching, and ten random sequence addition repetitions were used in the case of MP. The X2 tests used in PAUP* (Phylogenetic Analysis using Parsimony*) were used to analyze the sequences (Swofford and Sullivan 2003). The twelve valid Erythrobacter sequences were aligned using Clustal X for strains WH131 T and WH158 T , and phylogenetic trees were constructed using the maximum likelihood algorithms of MEGA X software (Kumar et al. 2018). The phylogenetic trees were supported by bootstrap for the resampling test with 100 replicates for the maximum likelihood algorithms.

Chemotaxonomy
Bacterial biomass was produced and collected after 7 days at 30 °C in a 250 mL flask containing 100 mL MB medium on a rotary shaker (160 revolutions per minute). The chemotaxonomic study was performed from freeze-dried biomass. Isoprenoid quinone production and analysis were conducted according to Minnikin (1984). Compounds were evaluated using high-performance liquid chromatography coupled with the diode array detector and mass spectrometer 488 Page 4 of 15 (HPLC-DAD-MS). The mobile phase consisted of 35% isopropanol + 1% water + 0.1% formic (solvent A) and 65% acetonitrile + 1% water + 0.1% formic acid (solvent B) and were used at a flow rate of 0.3 mL/min under isocratic conditions. Isoprenoid quinones were separated using a Waters ACQUITY UPLC BEH C18 column (2.1 × 50 mm, 1.7 m). Fatty acid extraction and methylation were carried out according to Sasser's procedure (Sasser 1990). Fatty acid methyl esters (FAME) were analyzed utilizing a gas chromatograph equipped with a flame ionization detector from Agilent (FID). Using a Macherey Nagel Optima 5 column, the methyl esters of fatty acids were extracted (5%phenyl, 95% dimethylpolysiloxane; 50 m length; 0.32 mm inner diameter; 0.25 m film thickness). Their retention periods were compared to standards (in-house reference standard) to identify specific fatty acid methyl esters.

Antimicrobial activity
Strains WHY3 T , WH131 T , and WH158 T were grown for 5 days at 30 ℃ on a shaker in 250-ml Erlenmeyer flasks that contained 100 mL of MB medium with 2% (v/v) XAD-2 polymeric resin (160 revolutions per minute). The separation of XAD-2 was accomplished by separating the resin with a paper filter from the media. Acetone was used to prepare the crude extract from the XAD-2. A rotary evaporator was used to dry the extract at a temperature of 40 °C. The dried extract was diluted in 1 mL methanol and evaluated for antimicrobial activity against a variety of the following bacteria:

Morphological, physiological and biochemical results
Cell size measurements are described in the description part for each strain. The electron microscopy images are available in supplementary file (Fig. S1).
The ideal temperature for growth for all three strains was 30 °C. Besides pH ranges, the tolerance of sodium chloride was recorded; the results of the biochemical property-based Api ZYM, Api 20NE, and Api 20E tests indicated positive activity for all strains WHY3 T , WH131 T , and WH158 T and related closes type strains for catalase, oxidase, phosphatase alkaline, leucin arylamidase, valine arylamidase; and negative for α-galactosidase, β-glucuronidase, N-acetyl-βglucosaminidase, α-mannosidase, α-fucosidase. Other comparisons for phenotypic characteristics and also Biolog Gen III system results between the isolated strains and closetrelated strains are observable in Table 1 and description. Sudan Black B indicates a preference for PHAs granules for strains WH131 T and WH158 T but not for WHY3 T .

16S rRNA gene analysis
According to the EZBioCloud server's results, strains WHY3 T , WH131 T , and WH158 T were most closely related to the following strain types: 97.6% to Winogradskyella flava SFD31 T , 96.7% to Winogradskyella echinorum KMM 6211 T for strain WHY3 T ; 99.1% Erythrobacter longus OCh101 T , 98.5% Erythrobacter insulae JBTF-M21 T for strain WH131 T ; 99.1% Erythrobacter insulae JBTF-M21 T , 98.6% Erythrobacter longus OCh101 T for strain WH158 T . 16S rRNA gene results of strain WHY3 T from phylogenetic dendrogram demonstrated proximity of 96.5% to Winogradskyella ouciana ZXX205 T . Phylogenetic trees based on 16S rRNA gene sequences of strains WHY3 T , WH131 T , and WH158 T and its closely related type strains are shown in Fig. 1 and Fig. 2. It shows that strain WHY3 T formed a highly supported cluster with Winogradskyella species (W. ouciana ZXX205 T and W. flava SFD31 T ). Furthermore, strain WHY3 T formed a well-supported branch alongside W. ouciana ZXX205 T ; and also strains WH131 T and WH158 T formed a highly supported cluster with Erythrobacter species (E. rubeus KMU-140 T , E. longus OCh101 T and E. insulae JBTF-M21 T ); strains WH131 T and WH158 T formed a branch alongside E. longus OCh101 T and E. insulae JBTF-M21 T , respectively.

Chemotaxonomic characterization
The major cellular fatty acids and polar lipids for strain WHY3 T were C 15:0 , anteiso-C 15:1 ω7c, iso-C 15:0 , C 16:1 ω7c, phosphatidylethanolamine, an unknown glycolipid, six unidentified aminolipids, and four unidentified lipids. The major cellular fatty acids and polar lipids for strains WH131 T and WH158 T were C 14:0 2-OH, t 18:1 ω12, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, two unknown glycolipids, sphingoglycolipids, two phosphatidylcholines, four unidentified lipids, and two unidentified aminolipids for strains WH131 T and C 17:0, and C 18:1 ω7c, diphosphatidylglycerols, phosphatidylethanolamine, phosphatidylglycerol, unknown glycolipids, sphingoglycolipids, phosphatidylcholine, four unidentified lipids, two unidentified aminolipids for strain WH158 T (see Fig.  S2 and Table S2). In all three, unknown fatty acids are the dominant constituents. The fatty acid iso-C 15:1 G is reported as one of the dominant ones in W. flava KCTC 52348 T and W. ouciana ZXX205 T , but it was absent in strain WHY3 T as well as anteiso-C 15:1 ω7c and C 16:1 ω7c which are present in strain WHY3 T but not in W. flava KCTC 52348 T and W. ouciana ZXX205 T . The fatty acid iso-C 18:0 is only reported in Erythrobacter rubeus KMU-140 T . C 18:1 ω7c is the largest amount in strain WH158 T , E. insulae JBTF-M21 T , and E. rubeus KMU-140 T but was absent in strains WH131 T , E. longus DSM 6997 T , and E. litoralis DSM8509 T . The fatty acid fatty t 18:1 ω12 represents the largest amount in strain WH131 T , E. longus DSM 6997 T and E. litoralis DSM8509 T . C 17:1 ω6c is reported as the major fatty acid in just E. insulae JBTF-M21 T (see Fig. S2 and Table S2).

Genomic characteristics and phylogenomic analysis
Only one 16S rRNA gene sequence was discovered in the whole-genome data of strains WHY3 T , WH131 T , and    WH158 T , showing that the genomic data were not contaminated by other species. The draft assembled genome sequence of strain WHY3 T comprised of the following: 3,532,486 bp with a G + C content of 34.4%. The genome included 3276 genes comprising 3194 protein-coding genes, 48 tRNA genes, 3 rRNA genes, and 4 non-coding RNA.
In parallel, strains WH131 T and WH158 T had 3,153,164 bp and 2,586,581 bp with a G + C content of 59.7% and 56.6%, respectively. The genome included 3077 genes comprising 3011 protein-coding genes, 44 tRNA genes, 3 rRNA genes, and 4 non-coding RNA for strain WH131 T and 2506 genes comprising 2447 protein-coding genes, 41 tRNA genes, 3 rRNA genes, and 2 non-coding RNA for strain WH158 T . The phylogenomic tree (Fig. S3) shows that strain WHY3 T is well supported by Winogradskyella species with pseudobootstrap support values > 60% from 100 replications, with average branch support of 65.5%. The phylogenomic tree (Fig. S4) with average branch support of 40.3% shows that strain WH131 T was located in a cluster, although it was not well supported, with Pseudopontixanthobacter vadosimaris JL3514 T which is not the Erythrobacter species. However, the strain WH158 T formed a well-defined tight cluster with Erythrobacter insulae JBTF-M21 T , but it was relatively close to Parerythrobacter jejuensis JCM16677 T . Therefore, another investigation was undertaken using whole-proteome-based GBDP distances. As a consequence, a phylogenetic tree with average branch support of 94.5% was produced, which was more trustworthy than the nucleotide-based phylogenomic tree result (Fig.  S5) since strains WH131 T and WH158 T were classified in the same clade as other Erythrobacter species with a very high support score. In the whole-proteome-based phylogenetic tree, strains WH131 T and WH158 T were located in a very high-supported clade together with E. rubeus KMU-140 T and E. insulae JBTF-M21 T , respectively.
Additionally, as shown in Table S3 and Table S4, all of the type strains had an ANI value less than the species cutoff value of 95% and dDDH scores less than the threshold value of 70%, indicating that strains WHY3 T , WH131 T , and WH158 T can be distinguished from the other known available Winogradskyella and Erythrobacter species (Chun et al. 2018).
The genes related to bioremediation using KBase database for Winogradskyella luteol WHY3 T , Erythrobacter ani WH131 T , Erythrobacter crassostrea WH158 T , and closest type strains are reported in Table 3.
Mercury resistance in the environment might be due to the presence of merT and merP genes in Winogradskyella luteol WHY3 T and Winogradskyella ouciana ZXX205 T , as described as a different approach to mercury resistance and bioaccumulation by marine bacteria (Zhang et al. 2020).
The presence of phnA gene in strain Winogradskyella luteol WHY3 T and other nearest type strains emphasized the role of Winogradskyella for oxidation of anthracene and phenanthrene, although these genes were not detected in Erythrobacter ani WH131 T and Erythrobacter crassostrea WH158 T . The heavy metal resistance protein cobalt-zinc-cadmium czcD gene was found in Winogradskyella luteol WHY3 T , Erythrobacter ani WH131 T and Erythrobacter crassostrea WH158 T . A related gene for granulate polyhydroxyalkanoates (PHAs) was present in Erythrobacter ani WH131 T and Erythrobacter crassostrea WH158 T . The genes cusA, cusB, cusC, and protein B (related to Copper resistance genes) and the Nickel-cobalt-cadmium resistance protein genes nccX were reported just for Erythrobacter ani WH131 T , but not for Erythrobacter crassostrea WH158 T (Table 3).
Additionally, gene annotation using RAST analysis (https:// rast. nmpdr. org) predicted 3319, 3147, and 2564 coding sequences in the genome of strains WHY3 T , WH131 T and WH158 T , respectively. The dominant fraction of subsystem features for strain WHY3 T were amino acids and derivatives (169), Cofactors-Vitamins-Prosthetic Groups-Pigments (123), protein metabolism (139), carbohydrates (93), Fatty Acids-Lipids, and Isoprenoids (50). Other genes which were detected that have a role in the development process, were present as follows: virulence, disease, and defense (24), stress Response (20), and metabolism of Aromatic Compounds (9). For the protein metabolism genes, a significant percentage was for Protein biosynthesis. The dominant fraction of subsystem features for strains WH131 T and WH158 T were amino acids and derivatives (214,158),

Conclusion
This polyphasic study indicates that isolates WHY3 T , WH131 T , and WH158 T are new species belonging to the genus Winogradskyella and Erythrobacter. Based on our results, we propose the name Winogradskyella luteol sp. nov. for strain WHY3 T ; and Erythrobacter ani sp.nov. and Erythrobacter crassostrea sp.nov. for strains WH131 T and WH158 T , respectively. Environmental pollution is one of the most serious issues that the twenty-first century is dealing with. Restoration and rehabilitation of contaminated sites have attracted a great deal of interest from the scientific community, with bioremediation as the main methods in such endeavors. Based on our genome analysis, all three strains, WHY3 T , WH131 T , and WH158 T , show they have the potential for bioremediation, as they contain certain important genes that have already been proven to be involved in bioremediation processes.
The type strain WHY3 T (= DSM 111804 T = NCCB 100833 T ) was isolated from Hemolymph of Pacific Oyster Crassostrea gigas, which was collected from Wilhelmshaven in Germany.
The GenBank/NCBI accession numbers for 16S rRNA Gene sequence and whole-genome sequence of strain WHY3 T are MW888983 and JAGSPD000000000, respectively.

Description of Erythrobacter ani sp.nov.
Erythrobacter ani (a'ni. L. gen. n. ani, of the anus, referring to anus area near the adductor muscle in Crassostrea gigas).
The type strain WH131 T (= DSM 112099 T = NCCB 100824 T ) was isolated from Hemolymph of Pacific Oyster Crassostrea gigas, which was collected from Wilhelmshaven in Germany.
The GenBank/NCBI accession numbers for 16S rRNA Gene sequence and whole-genome sequence of strain WH131 T are MW888981 and JAGSPB000000000, respectively.
The type strain WH158 T (= DSM 112102 T = NCCB 100877 T ) was isolated from Hemolymph of Pacific Oyster Crassostrea gigas, which was collected from Wilhelmshaven in Germany.
The GenBank/NCBI accession numbers for 16S rRNA Gene sequence and whole-genome sequence of strain WH158 T are MW888982 and JAGSPC000000000, respectively.
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