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 WHY3T, WH131Tand WH158T) 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.

Materials and methods

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 WHY3T, WH131T, and WH158T) 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.

Morphological, physiological and biochemical studies

Cells were grown on MB (Difco marine broth 2216) media for 3 days at 30 °C followed by morphological observations, including motility, using a light microscope (Zeiss Axio Sc pie. A1 microscope). Cells grown in MB media for 2 days at 30 °C were fixed with aldehydes (final concentrations: 5% formaldehyde and 2% glutaraldehyde), dehydrated in a gradient series of acetone, critical point dried, and coated with gold–palladium according to a previously published protocol (Landwehr et al. 2018). Images were acquired at various magnifications using a Zeiss Merlin field emission scanning electron microscope (FESEM) equipped with a 25:75% ratio of Everhart–Thornley SE and Inlens SEM detectors. The sodium chloride tolerance of strains WHY3T, WH131T, and WH158T was determined using the following NaCl (w/v) concentrations: 0%, 2.5%, 5.0%, 7.5%, 10%, 15%, 25%, and 30% using the method of Kutzner (1981). The growth of bacteria on solitary carbon and nitrogen sources was evaluated using Microlog GEN III plates (Biolog). Sudan black B staining was employed at a concentration of 3% (w/v in 70% ethanol) to identify polyhydroxyalkanoate granules (PHAs) (Legat et al. 2010). MA medium was used to assess growth over a variety of temperatures (4, 15, 20, 25, 30, 35, 40, and 45 °C) and pH values (pH 5, 6, 7, 8, 9, 10, and 11). Antibiotic susceptibility tests were performed on MA medium over 48 h with the following antibiotics: gentamycin (10 µg/mL), oxytetracycline (10 µg/mL), thiostrepton (50 µg/mL), trimethoprim (50 µg/mL), ampicillin (10 µg/mL), chloramphenicol (30 µg/mL), polymyxin (50 µg/ml), spectinomycin (50 µg/mL), kanamycin (30 µg/mL), cephalosporin (50 µg/mL), fusidic acid (50 µg/mL), bacitracin (50 µg/mL), erythromycin (15 µg/mL), and tetracycline (50 µg/mL).Table S1. Api Zym (Humble et al. 1977), Api 20NE, Api 20E (bioMe'rieux) (O'Hara et al. 1992), and GEN III microplate (Biolog) were used in the biochemical investigation.

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′–AGAGTTTGATCMTGGCTCAG–3′) and 1492R (5′–TACGGYTACCTTGTTACGACTT–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 WHY3T (1343 bp), WH131T (1443 bp), and WH158T (1486 bp) were almost completely sequenced and submitted to GenBank under the accession number MW888983, MW888981, and MW888982, respectively. The phylogenetically closest strains of WHY3T, WH131T, and WH158T were determined based on 16S rRNA gene sequence similarity using the EZBioCloud system (https://www.ezbiocloud.net/) (Yoon et al. 2017a). Phylogenetic analysis of the 16S rRNA gene of strains WHY3T 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 WH131T and WH158T, 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 (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.

Whole-genome analysis

Illumina's next-generation sequencing technology using MiSeq 600 cycle v3 was employed for whole-genome sequencing, and Unicycler was applied for genome de novo assembly (Wick et al. 2017). To estimate the purity of the 16S rRNA gene in whole-genome data, the ContEst16S technique was utilized (https://www.ezbiocloud.net/tools/contest16s) (Lee et al. 2017a). To accomplish automated genome annotation, the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) was employed (Tatusova et al. 2016). In addition, the draft genome assembly was submitted to the RAST (Rapid Annotation Using Subsystem Technology) database for metabolic reconstruction study (https://rast.nmpdr.org/) (Aziz et al. 2008). A phylogenomic tree was generated using the whole-genome sequence of strains WHY3T, WH131T, and WH158T and their closest phylogenetic relatives using the Type (Strain) Genome Server (TYGS) (Meier-Kolthoff et al. 2022). The Ezbiocloud and NCBI databases were used to extract whole-genome sequences of Winogradskyella flava KCTC 52348T, Winogradskyella ouciana ZXX205T, Winogradskyella echinorum KCTC 22026T, Erythrobacter insulae JBTF-M21T, Erythrobacter rubeus KMU-140T, Erythrobacter longus DSM 6997T, Erythrobacter litoralis DSM8509T, Pseudopontixanthobacter vadosimaris JL3514T and Parerythrobacter jejuensis JCM 16677T. Strains WHY3T, WH131T, and WH158T genome sequences were submitted to the Type (Strain) Genome Server (TYGS) (https://tygs.dsmz.de; accessed on 05 March 2022). All pairwise comparisons for phylogenomic inference were performed using Genome BLAST Distance Phylogeny (GBDP), and precise intergenomic distances were obtained using the 'trimming' process and distance formula d5 (Meier-Kolthoff et al. 2013). One hundred distance replicates were considered each. Using the specified settings, we created digital DDH (dDDH) values and confidence intervals using the Genome-to-Genome Distance Calculator (GGDC 3.0) (Meier-Kolthoff et al. 2022). Intergenomic distances and branch support from FASTME 2.1.6.1 were used to build a balanced minimal evolution tree, which included subtree pruning and regrafting postprocessing (SPR) (Lefort et al. 2015). The tree's branch support was calculated using 100 pseudobootstrap replications. The average nucleotide identity (ANI), genome size, and guanine and cytosine (G + C) content were determined using the OrthoANIu method (Yoon et al. 2017b) (https://www.ezbiocloud.net/tools/ani). To support the classification of strains WH131T and WH158T in the appropriate genus, the average amino acid identity (AAI) value (http://enve-omics.ce.gatech.edu/aai/index) and the percentage of conserved proteins (POCP) value (https://github.com/2015qyliang/POCP) were calculated (Qin et al. 2014). The RAST algorithm v1.073 from the KBase database https://narrative.kbase.us/ genes was used in the field of genome mining (The genome features were functionally annotated using the following algorithms: Kmers V2; Kmers V1; and protein similarity). The most important genes involving bioremediation from isolated strains of the genus Winogradskyella and Erythrobacter were annotated as follows: the gene responsible for converting the toxic form of mercury to the nontoxic form and uptake of mercury via membranes which carried the Hg(II) into the cytoplasm (merA, merB, MerC, MerE, MerF, MerT, MerP) (Sone et al. 2013; Zhang et al. 2020); polyaromatic hydrocarbons (PAHs) are abundant in nature and are a significant source of environmental concern owing to their persistence, toxicity, mutagenicity, and carcinogenicity (Cerniglia 1993); anthracene and phenanthrene may be oxidized by the PhnA dioxygenase as carbon and energy source (Kasai et al. 2003); polyhydroxyalkanoic acid (PHAs) are stored inside cells as carbon and energy reserves (Takahashi et al. 2017); degradation of phenol (dmpN) (Selvaratnam et al. 1997); chromate resistance genes (chrB)(Aguilar-Barajas et al. 2008); heavy metal resistance protein cobalt–zinc–cadmium (czcA, czcD) (Abdelatey et al. 2011); nickel–cobalt–cadmium resistance protein (nccA, nccX) (Lee et al. 2019); copper resistance (cusA, cusB, cusC, cusF, Copper resistance protein B) (see Table 3 for details) (Bondarczuk and Piotrowska-Seget 2013).

The draft genome of strains WHY3T, WH131T, and WH158T was submitted to NCBI/GenBank with the accession number (JAGSPD000000000), (JAGSPB000000000), and (JAGSPC000000000), respectively.

Antimicrobial activity

Strains WHY3T, WH131T, and WH158T 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: Citrobacter freundii DSM 30,039, Staphylococcus aureus Newman, Escherichia coli wild type BW25113, Escherichia coli acrB JW25113, Mycobacterium smegmatis ATCC 700,084, Pseudomonas aeruginosa DSM 19,882, Acinetobacter baumannii DSM 30,008, Bacillus subtilis DSM 10, Mucor hiemalis DSM 2656, Wickerhamomyces anomalus DSM 6766, and Candida albicans DSM 1665. The serial dilution procedure was applied using 96-well microplates in accordance with Khosravi Babadi et al. (2021).

Result and discussion

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 WHY3T, WH131T, and WH158T and related closes type strains for catalase, oxidase, phosphatase alkaline, leucin arylamidase, valine arylamidase; and negative for α-galactosidase, β-glucuronidase, N-acetyl-β-glucosaminidase, α-mannosidase, α-fucosidase. Other comparisons for phenotypic characteristics and also Biolog Gen III system results between the isolated strains and closet-related strains are observable in Table 1 and description. Sudan Black B indicates a preference for PHAs granules for strains WH131T and WH158T but not for WHY3T.

Table 1 Comparison of phenotypic characteristics that distinguish strains WHY3T, WH131T, and WH158T from the most closely related type strains
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16S rRNA gene analysis

According to the EZBioCloud server's results, strains WHY3T, WH131T, and WH158T were most closely related to the following strain types: 97.6% to Winogradskyella flava SFD31T, 96.7% to Winogradskyella echinorum KMM 6211T for strain WHY3T; 99.1% Erythrobacter longus OCh101T, 98.5% Erythrobacter insulae JBTF-M21T for strain WH131T; 99.1% Erythrobacter insulae JBTF-M21T, 98.6% Erythrobacter longus OCh101T for strain WH158T. 16S rRNA gene results of strain WHY3T from phylogenetic dendrogram demonstrated proximity of 96.5% to Winogradskyella ouciana ZXX205T. Phylogenetic trees based on 16S rRNA gene sequences of strains WHY3T, WH131T, and WH158T and its closely related type strains are shown in Fig. 1 and Fig. 2. It shows that strain WHY3T formed a highly supported cluster with Winogradskyella species (W. ouciana ZXX205T and W. flava SFD31T). Furthermore, strain WHY3T formed a well-supported branch alongside W. ouciana ZXX205T; and also strains WH131T and WH158T formed a highly supported cluster with Erythrobacter species (E. rubeus KMU-140T, E. longus OCh101Tand E. insulae JBTF-M21T); strains WH131T and WH158T formed a branch alongside E. longus OCh101T and E. insulae JBTF-M21T, respectively.

Fig. 1
figure 1

ML tree of strain WHY3T and its closely related type strains inferred under the GTR + GAMMA model and rooted by midpoint-rooting. The numbers above the branches are support values (above 60%) from ML (left) and MP (right) bootstrapping. The ML and MP bootstrapping average support were 26.12% and 42.40%, respectively

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Fig. 2
figure 2

Maximum Likelihood method tree for strains WH131T and WH158T, based on 16S rRNA gene sequences. Bootstrap values are expressed as percentages of 100 replications. Bar, 0.010 substitutions per nucleotide position. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 14 nucleotide sequences. There were a total of 1493 positions in the final data

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Chemotaxonomic characterization

The major cellular fatty acids and polar lipids for strain WHY3T were C15:0, anteiso-C15:1 ω7c, iso-C15:0, C16:1ω7c, phosphatidylethanolamine, an unknown glycolipid, six unidentified aminolipids, and four unidentified lipids. The major cellular fatty acids and polar lipids for strains WH131T and WH158T were C14:02-OH, t18:1ω12, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, two unknown glycolipids, sphingoglycolipids, two phosphatidylcholines, four unidentified lipids, and two unidentified aminolipids for strains WH131T and C17:0, and C18:1ω7c, diphosphatidylglycerols, phosphatidylethanolamine, phosphatidylglycerol, unknown glycolipids, sphingoglycolipids, phosphatidylcholine, four unidentified lipids, two unidentified aminolipids for strain WH158T (see Fig. S2 and Table S2). In all three, unknown fatty acids are the dominant constituents. The fatty acid iso-C15:1 G is reported as one of the dominant ones in W. flava KCTC 52348T and W. ouciana ZXX205T, but it was absent in strain WHY3T as well as anteiso-C15:1 ω7c and C16:1ω7c which are present in strain WHY3T but not in W. flava KCTC 52348T and W. ouciana ZXX205T. The fatty acid iso-C18:0 is only reported in Erythrobacter rubeus KMU-140T. C18:1ω7c is the largest amount in strain WH158T, E. insulae JBTF-M21T, and E. rubeus KMU-140T but was absent in strains WH131T, E. longus DSM 6997T, and E. litoralis DSM8509T. The fatty acid fatty t18:1ω12 represents the largest amount in strain WH131T, E. longus DSM 6997T and E. litoralis DSM8509T. C17:1ω6c is reported as the major fatty acid in just E. insulae JBTF-M21T (see Fig. S2 and Table S2).

Menaquinone-6 (MK-6) was found to be the major respiratory quinone in strain WHY3T. Ubiquinone-10 (Q-10) was detected in strains WH131T and WH158T.

Genomic characteristics and phylogenomic analysis

Only one 16S rRNA gene sequence was discovered in the whole-genome data of strains WHY3T, WH131T, and WH158T, showing that the genomic data were not contaminated by other species. The draft assembled genome sequence of strain WHY3T 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 WH131T and WH158T 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 WH131T and 2506 genes comprising 2447 protein-coding genes, 41 tRNA genes, 3 rRNA genes, and 2 non-coding RNA for strain WH158T. The phylogenomic tree (Fig. S3) shows that strain WHY3T is well supported by Winogradskyella species with pseudo-bootstrap 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 WH131T was located in a cluster, although it was not well supported, with Pseudopontixanthobacter vadosimaris JL3514T which is not the Erythrobacter species. However, the strain WH158T formed a well-defined tight cluster with Erythrobacter insulae JBTF-M21T, but it was relatively close to Parerythrobacter jejuensis JCM16677T. 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 WH131T and WH158T were classified in the same clade as other Erythrobacter species with a very high support score. In the whole-proteome-based phylogenetic tree, strains WH131T and WH158T were located in a very high-supported clade together with E. rubeus KMU-140T and E. insulae JBTF-M21T, respectively.

An additional study was conducted using genomic data to identify the genus classification, using the average amino acid identity (AAI) (Rodriguez-R and Konstantinidis, 2014) and the percentage of conserved protein (POCP) values. The POCP values and AAI values between the genomes of strains WH131T and WH158T against Erythrobacter insulae JBTF-M21T, Erythrobacter rubeus KMU-140T, Erythrobacter longus DSM 6997T, Erythrobacter litoralis DSM8509T, Pseudopontixanthobacter vadosimaris JL3514T, Parerythrobacter jejuensis JCM 16677T are shown in Table 2 with 81.9% (POCP) and 87.6% (AAI) as the highest percentages against Erythrobacter rubeus KMU-140T for strain WH131T and 73.0% (POCP) and 81.2% (AAI) as the highest percentages value against Erythrobacter insulae JBTF-M21T for strain WH158T (Qin et al. 2014; Rodriguez-R and Konstantinidis 2014). Thus, isolates WH131T and WH158T could be classified as Erythrobacter rather than Pseudopontixanthobacter or Parerythrobacter.

Table 2 Genome relatedness between the strains WH131T and WH158T, genus Erythrobacter, and other closet type strains according to the average amino acid identity (AAI) value and the percentage of conserved protein (POCP) values
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Additionally, as shown in Table S3 and Table S4, all of the type strains had an ANI value less than the species cut-off value of 95% and dDDH scores less than the threshold value of 70%, indicating that strains WHY3T, WH131T, and WH158T 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 WHY3T, Erythrobacter ani WH131T, Erythrobacter crassostrea WH158T, and closest type strains are reported in Table 3.

Table 3 Distribution of genes involved in bioremediation in 1, Winogradskyella luteola WHY3T; 2, Winogradskyella flava KCTC 52348T 3, Winogradskyella ouciana ZXX205T; 4, Winogradskyella echinorum KCTC 22026T; 5, Erythrobacter ani WH131T; 6, Erythrobacter crassostrea WH158T; 7, Erythrobacter insulae JBTF-M21T; 8, Erythrobacter rubeus KMU-140T; 9, Erythrobacter longus DSM 6997T; 10, Erythrobacter litoralis DSM8509T using https://narrative.kbase.us/ accessed on 04 April 2022
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Mercury resistance in the environment might be due to the presence of merT and merP genes in Winogradskyella luteol WHY3T and Winogradskyella ouciana ZXX205T, 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 WHY3T 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 WH131Tand Erythrobacter crassostrea WH158T. The heavy metal resistance protein cobalt–zinc–cadmium czcD gene was found in Winogradskyella luteol WHY3T, Erythrobacter ani WH131Tand Erythrobacter crassostrea WH158T. A related gene for granulate polyhydroxyalkanoates (PHAs) was present in Erythrobacter ani WH131Tand Erythrobacter crassostrea WH158T. 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 WH131T, but not for Erythrobacter crassostrea WH158T (Table 3).

Additionally, gene annotation using RAST analysis (https://rast.nmpdr.org) predicted 3319, 3147, and 2564 coding sequences in the genome of strains WHY3T, WH131Tand WH158T, respectively. The dominant fraction of subsystem features for strain WHY3T 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 WH131T and WH158T were amino acids and derivatives (214,158), protein metabolism (164, 84), carbohydrates (136,91), Cofactors–Vitamins–Prosthetic Groups–Pigments (110,94), Membrane Transport (100,42), Fatty Acids–Lipids, and Isoprenoids (80,65), Respiration (83,63) and Stress Response (51,31), respectively. The presence of protein and nucleoprotein secretion system gene (Type IV) was remarkable in strain WH131T as it is absent in strain WH158T (Fig. S6) (Table S5). The extract of strain WHY3T could moderately inhibit the growth of Staphylococcus aureus Newman, Candida albicans DSM 1665 and weak inhibited Bacillus subtilis DSM 10 and Wickerhamomyces anomalus DSM 6766; the extract of strains WH131T and WH158T showed no remarkable inhibition against the most tested microbes (Table 6).

Conclusion

This polyphasic study indicates that isolates WHY3T, WH131T, and WH158T are new species belonging to the genus Winogradskyella and Erythrobacter. Based on our results, we propose the name Winogradskyella luteol sp.nov. for strain WHY3T; and Erythrobacter ani sp.nov. and Erythrobacter crassostrea sp.nov. for strains WH131T and WH158T, 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, WHY3T, WH131T, and WH158T, show they have the potential for bioremediation, as they contain certain important genes that have already been proven to be involved in bioremediation processes.

Description of Winogradskyella luteola sp.nov.

Winogradskyella luteola (lu.te.o'la. L. fem. adj. luteola, light yellow).

Cells are Gram-negative-staining, motile by gliding, rod-shaped, aerobic, no-spore-form, devoid of flagella, 0.3–0.4 µm width and 0.8–2.1 µm in length. Colonies are yellow. Temperature range for growth is 5–40 (℃) pH spectrum for growth 6–9 and NaCl (optimum) for growth 2.5(%).

Positive for: catalase, oxidase, esterase (C4), esterase lipase (C8), phosphatase acid, naphtol-AS-BI-phosphohydrolase, acetoin, phosphatase alkaline, leucin arylamidase, valine arylamidase, assimilation of d-glucose, malic acid. Positive results (Biolog GEN III Micro Plate analysis) for glucuronamide, Gentiobiose, d-turanose, α-d-glucose, d-fucose, l-fucose, l-rhamnose, acetoacetic acid, acetic acid, l-malic acid, bromo-succinic acid, potassiumtellurite, sodium butyrate, sodium bromate, d-glucuronic acid, d-fructose-6-PO4, l-histidine, 1% NaCl, 4% NaCl and 8% NaCl, 1% sodium lactate, fusidic acid, d-serine, troleandomycin, rifamycin SV, minocycline, lincomycin, guanidine HCl, niaproof 4, vancomycin, nalidixic as well as susceptibility for chloramphenicol, thiostrepton, erythromycin, aztreonam. Major polar lipids are phosphatidylethanolamine (PE), unidentified glycolipid (GL), unidentified aminolipid (AL), and unidentified polar lipid (L). The predominant cellular fatty acids are C15:0, anteiso-C15:1 ω7c, iso-C15:0, C16:1ω7c. The menaquinone-6 (MK-6) is the major respiratory quinone. The DNA G + C content of type strain is 34.4%. Genome size of strain WHY3T indicates 3,53 Mbp.

The type strain WHY3T (= 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 WHY3T 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).

Gram-negative, no-spore-form, non-flagellated and coccoid, ovoid or rod-shaped cell, 0.2–0.3 µm width and 0.6–3.1 µm length: Colonies are yellowish-orange. Temperature range for growth is 20–35 (℃) pH spectrum for growth 6–9 and NaCl (optimum) for growth 2.5(%). Positive for: catalase, oxidase, esterase (C4), esterase lipase (C8), cystine arylamidase, trypsin, α –chymotrypsin, phosphatase acid, nitrate reduction, arginine dihydrolase, aesculin hedrolysis, tween 80 hydrolysis, phosphatase alkaline, leucin arylamidase, valine arylamidase, assimilation of d-glucose, d-mannose, d-mannitol, d-maltose, malic acid, phenylacetic acid. There are positive results (Biolog GEN III Micro Plate analysis) for glucuronamide, d-glucose-6-phosphate, d-fructose-6-phosphate, d-galacturonic acid, l-lactic acid, α-ketoglutaric acid, β-hydroxy-D, l-butyric acid, acetic acid, dextrin, d-maltose, d-trehalose, d-cellobiose, gentiobiose, sucrose, d-turanose, stachyose, d-raffinose, α- d-lactose, d-melibiose, β-methyl-d-glucoside, d-salicin, 1% NaCl, α-d-glucose, d-mannose, d-fructose, d-galactose, d-fucose, l-fucose, l-rhamnose, inosine, d-sorbitol, d-mannitol, d-aspartic acid, gelatin, glycyl-l-proline, l-glutamic acid, pectin, l-galactonic acid lactone, d-gluconic acid, d-glucuronic acid, mucic acid, quinic acid, methyl pyruvate, d-lactic acid methyl ester, d-malic acid, l-malic acid, bromosuccinic acid, p-Hydroxy phenylacetic acid, tween 40, α-ketobutyric acid, formic acid, potassium tellurite, aztreonam, sodium butyrate. The other substrates were inactive. Antibiotic susceptibility for gentamycin, kanamycin, chloramphenicol, spectinomycin, fusidic acid, thiostrepton, erythromycin and tetracycline. Major polar lipids are diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylglycerol (PG), sphingoglycolipid (SGL), unidentified glycolipid (GL), unidentified aminolipid (AL), and unidentified polar lipid (L). The predominant cellular fatty acids are C14:02-OH, t18:1ω12. The ubiquinone-10 (Q-10) is the predominant isoprenoid quinone. The DNA G + C content of type strain is 59.7%. Genome size of strain WHY3T indicates 3,15 Mbp.

The type strain WH131T (= DSM 112099T = NCCB 100824T) 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 WH131T are MW888981 and JAGSPB000000000, respectively.

Description of Erythrobacter crassostreae sp.nov.

Erythrobacter crassostreae (crass.os'tre.ae. N.L. gen. n. crassostreae, referring to the giant oyster Crassostrea gigas).

Gram-negative, no-spore-form, non-flagellated, and coccoid, ovoid or rod-shaped cell, 0.2–0.4 µm width and 0.5–2.4 µm length. Colonies are orange. Temperature range for growth is 20–35 (℃) pH spectrum for growth 6–9 and NaCl (optimum) for growth 2.5(%). Positive for: catalase, oxidase, esterase (C4), esterase lipase (C8), cystine arylamidase, trypsin, α-chymotrypsin, phosphatase acid, lipase (C14), Naphtol-AS-BI-phosphohydrolase, arginine dihydrolase, aesculin hedrolysis, phosphatase alkaline, leucin arylamidase, valine arylamidase, phosphatase alkaline, leucin arylamidase, valine arylamidase, assimilation of d-glucose, d-mannose, d-mannitol, d-maltose, phenylacetic acid. There are positive results (Biolog GEN III Micro Plate analysis) for dextrin, d-maltose, d-trehalose, d-cellobiose, gentiobiose, sucrose, d-turanose, stachyose, d-raffinose, α- d-lactose, d-melibiose, 1% NaCl, α-d-glucose, d-mannose, d-fructose, d-galactose, l-fucose, d-fucose, l-rhamnose, inosine, d-mannitol, d-glucose-6-phosphate, d-fructose-6-phosphate, glycyl-l-proline, l-glutamic acid, pectin, d-galacturonic acid, l-galactonic acid lactone, d-glucuronic acid, d-gluconic acid, glucuronamide, mucic acid, quinic acid, p-Hydroxy phenylacetic acid, methyl pyruvate, l-lactic acid, d-malic acid, l-malic acid, nalidixic acid, tween 40, α-ketobutyric acid, acetic acid, formic acid, aztreonam, sodium butyrate. The other substrates were inactive. Antibiotic susceptibility for gentamycin, kanamycin, chloramphenicol, spectinomycin, fusidic acid, thiostrepton, nalidixic acid, erythromycin, and tetracycline. Major polar lipids are diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylglycerol (PG), sphingoglycolipid (SGL), unidentified glycolipid (GL), unidentified aminolipid (AL), and unidentified polar lipid (L). The predominant cellular fatty acids are C17:0 and C18:1ω7c. The ubiquinone-10 (Q-10) is their predominant isoprenoid quinone. The DNA G + C content of type strain is 56.6%. The genome size of strain WHY3T indicates 2,58 Mbp.

The type strain WH158T (= 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 WH158T are MW888982 and JAGSPC000000000, respectively.