Introduction

The genus Thalassospira was established by López-López et al. (2002) and later emended by Liu et al. (2007) and Tsubouchi et al. (2014), belonging to the family Rhodospirillaceae within the class Alphaproteobacteria. At present, the genus contains 12 species with validly published and correct names according to the List of Prokaryotic Names with Standing in Nomenclature (LPSN) (www.bacterio.net, accessed on April 30, 2024) (Parte et al. 2020), and T. lucentensis is the type strain of the genus Thalassospira. Cells are Gram-staining negative, non-motile or motile using a polar flagellum, curved-to-spiral rod-shaped, aerobic to facultatively anaerobic, halophilic, and positive for catalase and variable to oxidase. The major cellular fatty acids are C18:1ω7c and C16:0, isoprenoid quinone is Q-9 or Q-10, and the range of genomic DNA G + C content is 46.0–62.0 mol% (Dong et al. 2018; López-López et al. 2002). From the Tara Oceans consortium metagenomic data, Thalassospira spp. make up to 0.1% of all marine bacteria (Sunagawa et al. 2015). The main habitat of the genus is seawater (Dong et al. 2018; Liu et al. 2016; López-López et al. 2002), marine sediments (Tsubouchi et al. 2014), and oil-contaminated water (Liu et al. 2007). Studies demonstrated the biodegradation ability of Thalassospira and suggested that these strains play an important role in marine contaminated ecosystems because of their potential in eliminating marine oil pollution, especially in polycyclic aromatic hydrocarbons degradation and polyvinyl-alcohol degradation (Nogi et al. 2014; Santisi et al. 2022; Wang et al. 2010). In addition, some species have potential beneficial properties of long-chain polyunsaturated fatty acids production for fish feed (Romano et al. 2020).

The genus Winogradskyella is a member of the family Flavobacteriaceae in the phylum Bacteroidota (Oren and Garrity 2021), which was first described by Nedashkovskaya et al. (2005), later emended by Ivanova et al. (2010), Yoon et al. (2011), Nedashkovskaya et al. (2012) and Begum et al. (2013). At present, the genus comprised 50 species with validly published names according to LPSN (accessed on April 30, 2024), and W. thalassocola is the type strain of the genus Winogradskyella. Members of the genus Winogradskyella have been isolated from different marine environments, such as alga specimens, seawater, marine sediment, coastal sediment, tidal flat, and coral (Bo et al. 2021; Lau et al. 2005; Yoon et al. 2011). Members of the genus Winogradskyella are heterotrophic, yellow or orange-pigmented, and Gram-stain-negative. All of them are mesophilic and slightly halophilic. The genomic DNA G + C content is less than 40.0 mol%, the major respiratory quinone is menaquinone 6 (MK-6), and phosphatidylethanolamine (PE) is the major polar lipid.

In this study, two strains FZY0004T and YYF002T were isolated from an agar-degrading coculture, which was obtained from seawater, and their taxonomic positions were clarified using a polyphasic approach. Based on these results, strains FZY0004T and YYF002T are proposed as novel species within the genera Thalassospira and Winogradskyella, respectively.

Material and methods

Isolation and culturing conditions

The seawater sample was collected from the intertidal zone (33°6′59″N, 121°51′9″E) of Yancheng City, China. Strains FZY0004T and YYF002T were isolated via a similar strategy as that for Marinilongibacter aquaticus YYF0007T (Zhang et al. 2022). The purified strains were preserved at − 80 °C as glycerol stocks (20%, v/v). Strains T. xianhensis CGMCC 1.6849T and T. profundimaris MCCC 1A00207T were obtained from the China General Microbiological Culture Collection Center (CGMCC) and the Marine Culture Collection of China (MCCC), respectively, and used as the experimental control of strain FZY0004T. Strain W. poriferorum JCM 12885T (Lau et al. 2005) was obtained from the Japan Collection of Microorganisms (JCM) and used as the experimental control of strain YYF002T.

16S rRNA gene sequencing and phylogenetic analysis

The genomic DNA was extracted and purified with an Ezup Column Bacteria Genomic DNA Purification Kit (Sangon Biotech, China). The 16S rRNA gene amplicons of the strains were obtained by PCR amplification. The PCR reaction included 2 × PCR Master (Sangon Biotech) and a universal bacterial primer pair (forward primer 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and reverse primer 1492R (5′-GGCTACCTTGTTACGACTT-3′) (Weisburg et al. 1991)). The sequence of the 16S rRNA gene was assembled using SeqMan software (DNASTAR) and submitted to the EzBioCloud (https://www.ezbiocloud.net/) (Yoon et al. 2017a) and NCBI (https://blast.ncbi.nlm.nih.gov/) database for alignment analysis with other taxa.

Evolutionary distances were computed using the Kimura 2-parameter method (Kimura 1980) for the Neighbor-Joining (NJ) algorithm (Saitou and Nei 1987) and a phylogenetic tree constructed with MEGA X software package (Kumar et al. 2018) after multiple sequence alignments. For comparison with the NJ phylogenetic tree, the Maximum Likelihood (ML) tree (Felsenstein 1981) and the Maximum-Parsimony (MP) tree (Kannan and Wheeler 2012) were constructed using the MEGA X Program. MEGA X software was used to create phylogenetic trees by bootstrap analysis with 1,000 replicates (Felsenstein 1981).The 16S rRNA gene sequences of Azorhizobium caulinodans ORS 571T (D11342) and Tamlana crocina HST1-43T (AM286230) were used as outgroups, respectively.

Genome sequencing and genome sequence analysis

Genome sequencing of strains FZY0004T and YYF002T was performed using a paired-end sequencing method with the Hiseq X platform (Illumina) at Personalbio Company, Shanghai, China. The genome of strain W. poriferorum JCM 12885T was also sequenced in this study because there is no genome sequence available for it. Sequence assembly was performed with SPAdes v3.13.0 (Nurk et al. 2013). The genomic DNA G + C content was calculated based on the whole-genome sequence. Genomic annotation was performed using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (https://www.ncbi.nlm.nih.gov/genome/annotation_prok/) to enhance the understanding of the genome. The genome sequence data of strains FZY0004T and YYF002T, along with their affiliated genera Thalassospira and Winogradskyella, were analysed using EasyCGTree version 4.2 (https://github.com/zdf1987/EasyCGTree4) under Windows operation system (OS) (Zhang et al. 2023) to clarify the phylogenetic relationship. This allowed the construction of a phylogenomic tree, providing insights into the evolutionary connections and taxonomic positioning of strains with its closely related species. The phylogenetic tree was constructed using the 120 ubiquitous single-copy protein-coding genes (Parks et al. 2018) from all the publicly available genomes of the genus Thalassospira or Winogradskyella, along with the outgroup. The average nucleotide identity (ANI) was estimated by using an ANI Calculator tool (https://www.ezbiocloud.net/) (Yoon et al. 2017b) and the average amino identity (AAI) was estimated by using the AAI calculator (http://enve-omics.ce.gatech.edu/aai/) (Qin et al. 2014). Digital DNA-DNA hybridization (dDDH) was assessed using the Genome­to-Genome Distance Calculator (GGDC) (https://ggdc.dsmz.de) (Meier-Kolthoff et al. 2013) with the recommended formula 2. Function gene enrichment was performed on the predicted coding genes of strains FZY0004T and YYF002T using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (www.genome.jp/kegg/) (Kanehisa et al. 2016).

Morphology, physiology, and biochemical analysis

A routine cultivation on the R2A plates at 28 °C until the late-exponential growth phase was employed to investigate the morphological and physiological properties of strains FZY0004T and YYF002T. Gram-staining was tested by using a Gram Staining kit (G1060, Solarbio, China). Growth conditions were tested at 28 °C for 3 days on R2A, tryptic soy agar (TSA), marine agar 2216 (MA), and Luria–Bertani agar (LB), which were supplemented with 2.5% sea salt except for MA. NaCl tolerance (0, 0.5, 1.0, 2.0, 3.0, 5.0, 6.0, 7.0, 10.0, and 12.0%, w/v) was tested at 28 °C with initial pH 7.0 on R2A medium. The pH range for growth (adjusted from pH 4.0–10.0 in increments of 0.5 pH unit) was tested with the pH buffer system described by Xu et al. (2005) by culturing the strains in R2A broth. The optimal temperature range required for growth (4, 10, 20, 24, 28, 37 and 42 °C) was tested at pH 7.0 on R2A plates. The growth of the strains cultured at 4 °C and 42 °C was observed after two weeks. The morphology of cells and size were examined by scanning electron microscopy (Regulus 8100, HITACHI, Japan). After 3 days of incubation at 28 °C, the cell diffusion in semisolid R2A containing 0.5% agar was observed to determine the motility of the cells. Oxidase activity was tested using an oxidase reagent kit (bioMérieux) according to the manufacturer’s instructions and catalase activity was tested by observing the production of bubbles with 3% (w/v) H2O2. Various biochemical tests, including hydrolysis of starch, hydrolysis of cellulose, urea, Tween 20, Tween 60, Tween 80, indole production, and H2S production. Tests for other physiological or biochemical characteristics were performed using API 20E, API ZYM (all from bioMérieux), and GEN III MicroPlates (BIOLOG, USA), according to the manufacturer’s instructions, except that the salinity of the media was adjusted to 2.5%. Type strains of taxonomically related species T. xianhensis CGMCC 1.6849T, T. profundimaris MCCC 1A00207T, and W. poriferorum JCM 12885T were also tested for biochemical and chemical taxonomic analyses to allow a more complete comparison.

Chemotaxonomic characterization

Strains FZY0004T, YYF002T, and their reference strains were cultured in R2A broth at 28 °C for 48 h. Cells were collected by centrifugation and washed with distilled water. For fatty acid analysis cells were streak (quaternary streaking) plated on TSA agar. Cellular fatty acids were saponified, methylated and extracted following the classical method of MIDI protocol (Sherlock Microbial Identification System, version 6.2B) (Sasser 1990). Quinones were extracted with a chloroform/methanol (2:1, v/v) mixture and analysed using high-performance lipid chromatography (HPLC) (HIiraishi et al. 1996). Polar lipids were extracted and identified by using two-dimensional thin-layer chromatography as the previously described method (Athayle et al. 1984).

Results and discussion

Phylogenetic analysis based on 16S rRNA gene sequences

The comparative analysis of 16S rRNA gene sequences from the EzBioCloud database revealed that strain FZY0004T (accession number, OQ714501) was closely related to T. povalilytica Zumi 95T (99.5%), T. australica NP3b2T (99.5%), T. profundimaris WP0211T (99.4%), T. xiamenensis M-5T (99.4%), T. xianhensis P-4T (99.4%), T. tipidiphila 1-1BT (99.3%), T. indica PB8BT (99.0%), T. lucentensis DSM 14000BT (98.7%), T. lohafexi 139Z-12T (98.7%), T. alkalitolerans MBE#61T (98.0%), T. mesophila MBE#74T (97.9%), and T. marina CSC3H3T (97.8%). Strain YYF002T (accession number, OQ714211) was closely related to W. poriferorum UST030701-295T (99.2%) and W. aquimaris DPG-24T (97.0%). The strains annotated in the EzBioCloud database with 16S rRNA gene similarity exceeding 97% are the same as those annotated in the NCBI database. Phylogenetic analysis using the NJ, ML, and MP algorithms showed that strain FZY0004T formed a phyletic lineage with T. australica NP3B2T, T. povalilytica Zumi 95T, T. profundimaris WP0211T, T. indica PB8BT and T. tipidiphila 1-1BT within the genus Thalassospira (Fig. S1). The results of phylogenetic analysis based on the NJ and MP method indicated that YYF002T formed a phyletic lineage with W. poriferorum UST030701-295T, and the corresponding ML trees showed similar topologies that W. poriferorum UST030701-295T was its closest neighbor, which supported the proposal that strains YYF002T belonged to the genus Winogradskyella (Fig. S2).

Genomic characteristic

The whole genome sequence size of FZY0004T was 4,982,113 bp. The N50 was 375,977 bp and the DNA G + C content was 54.5%. The genome of FZY0004T contained 4,573 genes in total and included 4,502 protein-coding genes, 3 rRNAs, 56 tRNAs, and 34 contigs. The draft genome sequence size of YYF002T was 3,524,355 bp, with an N50 of 437,365 bp and a DNA G + C content of 33.5%. The genome of YYF002T contained 3,166 genes in total and included 3,116 protein-coding genes, 3 rRNAs, 38 tRNAs, and 29 contigs. The draft genome sequence size of W. poriferorum JCM 12885T was 3,621,352 bp, with an N50 of 739,931 bp and a DNA G + C content of 33.5%. The genome of JCM 12885T contained 3,284 genes in total and included 3,228 protein-coding genes, 3 rRNAs, 42 tRNAs, and 17 contigs (Table S1). The number of genes associated with the KEGG functional pathways in strains FZY0004T and YYF002T and the reference type strains were shown in Tables S2 and S3.

In the phylogenomic bac120 tree, FZY0004T and T. povalilytica Zumi 95T made a separate clade (Fig. 1), as did YYF002T and W. poriferorum JCM 12885 T (Fig. 2), which is consistent with the results for the phylogenetic tree based on 16S rRNA gene sequences. The ANI values for FZY0004T and the reference strains T. profundimaris WP0211T and T. xianhensis MCCC 1A02616T were 85.0% and 79.3%, respectively, and those for YYF002T and the reference strain W. poriferorum JCM 12885 T was 88.2%. The AAI values for FZY0004T and the reference strains T. profundimaris WP0211T and T. xianhensis MCCC 1A02616T were 81.2% and 78.1%, respectively, and those for YYF002T and the reference strain W. poriferorum JCM 12885 T was 89.9%. The dDDH values for FZY0004T and the reference strains T. profundimaris WP0211T and T. xianhensis MCCC 1A02616T were 23.6% and 21.5%, respectively, and those for YYF002T and the reference strain W. poriferorum JCM 12885 T was 34.7%. All values were below the threshold for species delineation, which are 95%, 95%, and 70% for ANI, AAI, and dDDH, respectively (Meier-Kolthoff et al. 2013; Richter and Rossello-Mora 2009; Wayne et al. 1987). Detailed values of the two novel strains and their close species are given in Tables 1 and 2. As a result, a phylogenetic tree was conducted based on coding sequences of 120 protein clusters and revealed that FZY0004T and YYF002T represent members of the genera Thalassospira and Winogradskyella, respectively.

Fig. 1
figure 1

A maximum-likelihood tree based on the 120 ubiquitous single-copy protein-coding genes showing the phylogenetic relationship of FZY0004T in the genus Thalassospira. Bootstrap values based on 1000 replicates are shown at the branch points nodes. The RefSeq assembly accession number is indicated in the bracket. Azorhizobium caulinodans ORS 571T (GCF_0000105525.1) is used as an outgroup and hidden in the tree. Bar, 0.05 substitutions per nucleotide position

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Fig. 2
figure 2

A maximum-likelihood tree based on the 120 ubiquitous single-copy protein-coding genes showing the phylogenetic relationship of YYF002T in the genus Winogradskyella. Bootstrap values based on 1000 replicates are shown at the branch points nodes. The RefSeq assembly accession number is indicated in the bracket. The type strain of the genus Winogradskyella, W. thalassocola DSM 15363T (GCF_900099995.1) was included in the clade. Tamlana crocina HST1-43T (GCF_012037625.1) is used as an outgroup and hidden in the tree. Bar, 0.05 substitutions per nucleotide position

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Table 1 Comparative genomic characteristics of strain FZY0004T and phylogenetically related species of the genus Thalassospira
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Table 2 Comparative genomic characteristics of strain YYF002T and phylogenetically related species of the genus Winogradskyella
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Physiology and biochemical analysis

Cells of strains FZY0004T and YYF002T were observed to be Gram-stain-negative. SEM images of strain FZY0004T cell showed curved rods with 0.3–0.5 μm width and 1.0–1.8 μm length (Fig. 3a), YYF002T showed rod-shaped profile (0.3–0.4 μm wide and 1.5–1.8 μm long (Fig. 3b). Srains FZY0004T and YYF002T could grow on R2A, TSA and MA plates, with the best growth on R2A plates. Both strains FZY0004T and YYF002T were positive for oxidase and catalase activity. Results of strain FZY0004T and the reference type strains T. profundimaris MCCC 1A00207T and T. xianhensis CGMCC 1.6849 T in BIOLOG GEN III microtest were shown in Table S4. Results of strain YYF002T in BIOLOG GEN III microtest weren’t shown, as the positive control failed. The comparisons of phenotypic and biochemical characteristics of strains FZY0004T and YYF002T with those of the reference strains are described in detail in Tables 3 and 4.

Fig. 3
figure 3

Scanning electron micrograph of strains FZY0004T (a) and YYF002T (b). Cells were grown on R2A media at 28 °C for 2 days. Bar, 1.0 μm

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Table 3 Differential characteristics between strain FZY0004T and the reference strains
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Table 4 Differential characteristics between strain YYF002T and the reference strain
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Chemotaxonomic characterization

The major fatty acids (> 10%) of strain FZY0004T were summed feature 3 (C16:1 ω7c and/or C16:1 ω6c) (14.5%), C16:0 (22.2%) and summed feature 8 (C18:1 ω7c and/or C18:1 ω6c) (24.2%). C16:0 is the most plentiful fatty acid in the closely related species of the genus Thalassospira investigated in this study. The detailed fatty acid compositions are presented in Table S5. However, some quantitative differences in fatty acid composition were observed between FZY0004T and the other closely associated species of the genus Thalassospira. For example, FZY0004T contained 24.2% summed feature 8 (C18:1 ω7c and/or C18:1 ω6c), but T. profundimaris MCCC 1A00207T and T. xianhensis CGMCC 1.6849 T contained 12.1% and 29.7%, respectively. The major fatty acids (> 10%) of YYF002T were iso-C15:0 3-OH (12.0%) and iso-C15:1 G (29.3%), with iso-C15:0 (33.1%) being the most plentiful fatty acid in the closely related species of the genus Winogradskyella investigated in this study. The detailed fatty acid compositions are presented in Table S6. The polar lipids of strain FZY0004T were found to be phosphatidylethanolamine (PE), phosphatidylglycerol (PG), unidentified phospholipids (PL), and unidentified lipids (L), with the major polar lipids being PE and PG. Polar lipids of YYF002T included unidentified amino lipid (AL), phosphatidylethanolamine (PE), and unidentified lipids (L). The major polar lipids of YYF002T were AL and PE, which are also the major polar lipids found in members of the genus Winogradskyella. The detailed polar lipid profiles of the two novel strains are provided in Fig. S3. The predominant respiratory quinones of strain FZY0004T were identified as Q-9, whereas that detected in strain YYF002T was MK-6.

Taxonomic conclusion

In summary, the phylogenetic topologies and phenotypic and chemotaxonomic characteristics supported that strains FZY0004T and YYF002T represent novel species of the genera Thalassospira and Winogradskyella, respectively, for which the names Thalassospira aquimaris sp. nov. and Winogradskyella marincola sp. nov. are proposed, respectively.

Description of Thalassospira aquimaris sp. nov.

Thalassospira aquimaris (a.qui.ma'ris. L. fem. n. aqua water; L. neut. n. mare the sea; N.L. gen. n. aquimaris of the water of the sea).

Cells are Gram-stain-negative, aerobic, non-motile, and curved rods with a size of 1.0–1.8 µm long and 0.3–0.5 µm wide. Colonies are white coloured, circular, convex, and 0.3–0.5 mm in diameter after 2 days of cultivation at 28 °C on R2A agar. The optimum temperature and pH for growth are 28 °C (range 10–37 °C) and 7.0 (6.0–9.0), respectively. NaCl is not essential for growth (tolerate up to 10% w/v; optimum, 2–6% w/v). Positive for H2S production and Tween 80 hydrolysis, but negative for hydrolysis of starch, Tween 20, and Tween 60, and indole production. Catalase and oxidase are positive. The major fatty acids are summed feature 8 (C18:1 ω7c/C18:1 ω6c), C16:0, and summed feature 3 (C16:1 ω7c/C16:1 ω6c). The major respiratory quinone is Q-9 and the major polar lipids consist of phosphatidylethanolamine (PE), phosphatidylglycerol (PG), unidentified phospholipids (PL), and unidentified lipids (L).

The type strain, FZY0004T (= JCM 35895 T = MCCC 1K08380T), was isolated indirectly from seawater in the Yellow Sea, China. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain FZY0004T is OQ714501 and its draft genome was deposited in GenBank/EMBL/DDBJ under the accession number JARSBO000000000. The DNA G+C content of the type strain is 54.5% (by genome).

Description of Winogradskyella marincola sp. nov.

Winogradskyella marincola (mar.in'co.la. L. neut. n. mare, the sea; L. masc./fem. n. incola inhabitant; N.L. fem. n. marincola, inhabitant of the sea).

Cells are Gram-stain-negative, aerobic, non-motile, and rod-shaped and are 1.5–1.8 µm long and 0.3–0.4 µm wide. When grown on R2A agar at 28 °C for 2 days, colonies are circular, convex, glistening, viscid, translucent, yellowish, and 0.3–0.4 mm in diameter. Growth does not occur without sea salts. Cells grow at 10–37 °C (optimum, 28 °C), in the presence of 0.5–5% (w/v) NaCl (optimum, 2–4%) and at pH 5.0–9.0 (optimum, pH 7.5). Catalase and oxidase are positive. Cells hydrolyse Tween 20, Tween 60, and Tween 80, but do not hydrolyse starch, cellulose, and urease, and are negative for H2S and indole production. The major fatty acids are iso-C15:0, iso-C15:1 G, and iso-C15:0 3-OH. The major respiratory quinone is MK-6 and the major polar lipids consist of unidentified aminolipid (AL), phosphatidylethanolamine (PE), and several unidentified lipids (L).

The type strain, YYF002T (= JCM 35950 T = MCCC 1K08382T), was isolated indirectly from seawater in the Yellow Sea, China. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain YYF002T is OQ714211 and its draft genome was deposited in GenBank/EMBL/DDBJ under the accession number JARSBN000000000. The DNA G+C content of the type strain is 33.5% (by genome).

Emended description of Winogradskyella poriferorum (Lau et al. 2005)

The description is as given previously (Lau et al. 2005) with the following amendment. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain JCM 12885 T is AY848823 and its draft genome was deposited in GenBank/EMBL/DDBJ under the accession number JAZHOU010000000. The DNA G+C content of the type strain is 33.5% (by genome).