Introduction

Carotenoids are yellow, orange or red isoprene pigments from a wide range of sources, which can be synthesized by photosynthetic organisms (plants, cyanobacteria, anoxygenic phototrophic bacteria and aerobic anxoygenic phototrophic bacteria), non-photosynthetic microorganisms1,2. There are more than 600 carotenoids found naturally. Carotenoids have high commercial application value. At present, the carotenoids commonly used as animal feed and food additives on the market mainly include lutein, β-carotenenes, lycopene and astaxanthin3. Astaxanthin (3,3’-dihydroxy-β,β b-caroten-4,4’-dione) is a xanthophyll carotenoid which is a secondary metabolite responsible for a red fat-soluble pigment4. It is produced predominantly by marine microorganisms and animals5,6. Astaxanthin has several essential biological activities such as powerful antioxidant, anti-inflammatory, and antiapoptotic activities7,8. Natural astaxanthin is Generally Recognized as Safe (GRAS)9. It is widely used in the food, animal feed, nutraceutical and pharmaceutical industries.

The genus Echinicola was first described by Nedashkovskaya et al.10. The member of the genus Echinicola are characterized as Gram-negative, aerobic or facultatively anaerobic, rod-shaped and pigmented bacteria10. The genus is affiliated with the family Cyclobacteriaceae belonging to the class Cytophaia of the phylum Bacteroidetes. At the time of writing, the genus Echinicola accommodates 9 species with validly published names and 3 published but not valid species: Echinicola pacifica10, Echinicola vietnamensis11, Echinicola jeungdonensis12, Echinicola roses13, Echinicola sediminis14, Echinicola strongylocentroti15, Echinicola soli16, Echinicola arenosa17, Echinicola salinicaeni18 and ‘Echinicola shivajiensis’19. These Echinicola type strains were isolated from various hypersaline environments, including sea urchin10,15, sea water11,13,17, saline soil16,18, solar saltern12, coastal sediment14 and brackish water19.

Marine Bacteroidetes are commonly assumed to be specialized in degrading polysaccharides20 due to a great number and diversity of carbohydrate-active enzymes (CAZymes) in their genomes21. Echinicola rosea JL3085T genome harbors multi-gene polysaccharide utilization loci (PUL) systems involved in the degradation of pectin, xylan and arabinogalactan22. And a xylanase gene (xynT) from Echinicila rosea JL3085T was cloned and recombinantly expressed in Escherichia coli BL2123. The O-specific polysaccharides are isolated from the lipopolysaccharide of Echinicola vietnamensis KMM 6221T and Echinicola pacifica KMM 6172T that reveals the polysaccharide is built up of branched tetrasaccharide repeating units24,25. Therefore, it is suggested that Echinicola strains play an important role in the carbon cycle in the marine environment.

Strain SCS 3–6, a pink-pigmented strain was isolated from deep sea sediment of the South China Sea and found to belong to the genus Echinicola based on 16S rRNA gene sequences and its phenotypic characteristics. And the whole genome sequences were obtained for the newly strain. In addition, whole genome sequences of some type strains of Echinicola species are available in GenBank database, the genome sequences have not been used previously for taxonomic analysis or subjected to comparative genomic studies. Therefore, we used the available genome sequence of Echinicola species to investigate the taxonomic status of the newly isolated strains with the genus Echinicola and to identify the differences by genotypic characteristics. With genotypic analysis and the examination of its carotenoid production ability, strain SCS 3–6 is an astaxanthin-producing strain.

Materials and methods

Sampling sites, enrichment and isolation

Strain SCS 3–6 was isolated from deep-sea sediment sample collected from the South China Sea (depth of 1700 m, E 117°56.2877′, N 20°59.8047′). In enrichment experiments for isolating strain SCS 3–6, 1 g of sediment sample was enriched in 50 ml marine agar (Difco) for 72 h at 28 °C, 150 rpm. Then, 200 μl of enriched solution was transferred to fresh medium and was cultured at 28 °C, 150 rpm for 72 h. And this routine culturing was repeated three times. After, the supernatants of the enriched sample were serially diluted (10–5 to 10–7) with PBS buffer (KH2PO4 0.2 g, Na2HPO4·12H2O 2.9 g, NaCl 8 g, KCl 0.2 g, pH 7.0). 100 μl of each diluted sample was spread on marine agar plates and incubated at 28 °C for 48 h.

Morphological, physiological, and biochemical analysis

The morphological characteristics of the strains were investigated after 24 h of incubation on marine agar. The Gram reaction was examined according to Buck’s method26. Cell morphology was investigated using scanning electron microscope (SU8010, Hitachi, Japan) and transmission electron microscope (H-7650, Hitachi, Tokyo, Japan). Growth was observed at various temperatures (4, 15, 20, 25, 28, 30, 33, 37, 40, 45, and 50 °C) on marine agar. Tolerance to different NaCl concentrations (0–10%, in increments of 1%, w/v, NaCl) and pH range (pH 4.0–11.0, at intervals of 1 unit) were performed at 28 °C, for 7 days. Anaerobic growth was tested in an MGCAnaeroPouch-Anaero (Mitsubishi, Tokyo, Japan) at 28 °C for 7 days on marine agar plates. Catalase and oxidase activities were investigated in 3% (v/v) H2O2 and using commercial strips (Huankai, Guangzhou, China) according to the manufacturer’s instruction, respectively. Additional enzyme activities and carbon source utilization assays were examined by using API 20NE, API ZYM (bioMerieux, Marcy-l′Etoile, French) and Biolog plates kits (Hayward, CA, USA), respectively, following the manufacturer’s instruction.

Chemotaxonomic analysis

For analysis of the chemotaxonomic features of strain SCS 3–6, a series of experiments27 were carried out to determine the content of the respiratory quinones, polar lipids, and fatty acids of closely related type strains (Echinicola shivajiensis JCM 17847T and Echinicola sediminis KCTC 52495T) and SCS 3–6 with cell biomass obtained from cultures grown in marine agar (Difco) for 2 days at 28 °C. Respiratory quinones were extracted from freeze-dried cells (100 mg) with chloroform/methanol (2:1) and analyzed via the HPLC system28. Polar lipids were extracted by using a chloroform/methanol/water system29 and separated by two-dimensional TLC. The plate dotted with the sample was subjected to two-dimensional development, with the first solvent of chloroform/methanol/water (65:25:4, by vol.) followed by the second solvent of chloroform/methanol/acetic acid/water (85:12:15:4, by vol.). The polar lipids were identified by spraying with phosphomolybdate, ninhydrin, Dragendorff’s reagents, molybdenum blue, 1-methylnaphthol, respectively. For cellular fatty acid analysis, fatty acids were saponified, methylated, and extracted according to the Microbial Identification System (MIDI) protocol. The fatty acid methyl eaters, analysed with a gas chromatograph (7890, Hewlett Packard), were identified by the Microbial Identification software package, based on the Sherlock Aerobic Bacterial Database (TSBA6)30.

Sequencing and phylogenetic analyses

Amplification of the 16S rRNA gene was performed by PCR with two primers: 27F and 1492R31. The amplified gene was ligated into the pJET1.2/Blunt Vector (Thermo Scientific, Waltham, MA, USA) and sequenced by Sangon Biotech (Shanghai, China). The 16S rRNA gene sequences used EzBioCloud’s Identify services32 to get sequences informations. The phylogenetic tree was constructed using the ClustalW algorithm from the MEGA 1133 software package using the neighbor-joining (NJ) and maximun likelihood (ML) methods followed by bootstrap analysis with 1000 bootstrap replications34.

Genome DNA sequencing, de novo assembly, annotation and genomes comparison

For genomic DNA extraction and sequencing, strain SCS 3–6 was inoculated from glycerol stocks in marine agar, and grown for 24 h at 28 °C, 200 rpm. Then, bacteria were washed in 1 × PBS and collected by centrifugation at 5000 rpm for 10 min at 4 °C. The genome of SCS 3–6 was extracted and sequence by Majorbio Bio-pharm Technology Co., Ltd (Shanghai, Chain) on PacBio and illumina Hiseq × 10 platform. A high-quality data set with a corresponding sequencing depth of 100-fold was generated. The scan map of the bacterial genome is created using SOAPdenovo235 and the complete map of the bacterial genome is assembled using Canu and SPAdes36. Glimmer and GeneMarkS37 were used to predict coding sequences (CDS) and plasmid genes, respectively. tRNA and rRNA were predicted using tRNAscan-SE v2.0 and Barrnap, respectively. Function annotation of SCS 3–6 was obtained from Non-Redundant Protein (NR), Swiss-Prot, Pfam, Clusters of Orthologous Group (COG), Gene Ontology and Kyoto Encyclopedia of Genes databases using BLASTp and the same BLAST thresholds38. Additionally, the CAZymes were identified, classified and annotated using CAZy database39. The gene clusters of secondary metabolites were identified by antiSMASH program40.

The average nucleotide identity (ANI) was calculated using the BLAST (ANIb) and MUMmer (ANIm) algorithms (http://jspecies.ribohost.com/jspeciesws/) 41 DNA–DNA hybridization (DDH) was calculated according to the method described by Meier-Kolthoff et al.42. The BPGA pipeline43 was used to perform model extrapolations of the Echinicola pangenome/core genome by applying default parameters.

The assay of its carotenoid production ability

The strains were inoculated into 100 mL marine agar, cultured at 28 °C for 72 h, centrifuged, and the bacteria were collected. Add 3 mL sterile water to the tube and wash the bacteria. Transfer the bacteria to a 15 mL centrifuge tube. Centrifuge at 4 °C, 8000×g for 10 min, then discard supernatant. The bacteria were resuspended with 2 mL acetone solution and extracted by shock for 10 min. After centrifugation at 8000×g for 10 min, the supernatant extract was transferred to a new tube. 2 mL ethyl acetate solution was added to the original tube, and the extraction was continued with oscillation for 10 min. After centrifugation at 8000×g for 10 min, the supernatant extract was mixed with the previous extract, and 3 mL sterile water was added to it, and the mixture was shaken and mixed. Centrifuge 8000×g for 10 min to delaminate the liquid and draw the upper liquid into a new EP tube. The ethyl acetate was dried to obtain powder and was redissolved with methanol. Over 0.22 µm filter membrane, used for High-pressure liquid chromatography (Shimadzu HPLC LC-20AT) analysis and detection. The tests were by C18 column with a mobile phase of methanol–acetonitrile–water (volume ratio being 80:15:5). UV detection was performed at 478 nm.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Results and discussion

Phylogenetic characteristics

The search of 16S rRNA gene sequence against the EzTaxon database revealed that strain SCS 3–6 belongs to Bacteroidetes phylum. The nearly complete 16S rRNA gene sequence (1518 bp) of strain SCS 3–6 was compared with other strains with the top 30 sequence similarity using phylogenetic tree analysis. Strain SCS 3–6 was 98.29% similar to Echinicola arenosa CAU 1574T, 97.98% with Echinicola shivajiensis AK12T, 96.97% with Echinicola sediminis 001-Na2T, and 96.22% with Echinicola jeungdonensis HMD3054T. The other species of the genus Echinicola showed < 96% sequence similarities with strain SCS 3–6. The NJ phylogenetic tree revealed strain SCS 3–6 clustered with members of the genus Echinicola and formed a monophyletic clade with Echinicola arenosa CAU 1574T (Fig. 1).

Figure 1
figure 1

Phylogenetic tree constructed with 16S rRNA gene by the NJ algorithm. The tree was rooted using Flavobacterium aquatike LMG4008T as the outgroup. Bootstrap values (> T50%) are shown at the nodes. Closed circles (·) represent branches recovered in maximum likelihood (ML) tree. The phylogenetic tree was constructed using the ClustalW algorithm from the MEGA 11 software (MEGA 11: https://www.megasoftware.net).

Full size image

Phenotypic properties

Cells of strain SCS 3–6 were Gram-stain negative, non-motile by gliding, and rod-shaped (0.2–0.3 μm in width and 1.5–2.0 μm in length) (Fig. 2). Colonies of this strain were circular, convex and pink pigmented after 2–3 days of incubation 30 °C on marine agar plates. Strain SCS 3–6 were facultative anaerobes. The strain SCS 3–6 was capable of growth at temperatures between 10 and 45 °C and the strain grew well at pH values between 5.0 and 9.0. The strain SCS 3–6 was tolerant to 10% (w/v) NaCl.

Figure 2
figure 2

Morphology of Echinicola marina sp.nov. Scanning electron microscope (SEM) images of strain SCS 3–6 (left) and transmission electron microscope (TEM) images of strain SCS 3–6 (right).

Full size image

The novel isolate can use various carbohydrates for growth. The following carbohydrates were utilized: d-maltose, d-trehalose, d-cellobiose, gentiobiose, sucrose, d-turanose, α-d-lactose, d-melibiose, d-salicin, N-acetyl neuraminic, α-d-glucose, d-mannose, d-fructose and d-galactose. Catalase and oxidase reactions were both positive. Strain SCS 3–6 was positive for the hydrolysis of gelatin and casein, but negative for starch and tween 80. Biochemical characteristics that differ between strain SCS 3–6, Echinicola shivajiensis AK12T and Echinicola sediminis 001-Na2T are shown in Table 1.

Table 1 Differential physiological characteristics between strain SCS 3–6 and closely related type strains.
Full size table

The major cellular fatty acids were iso-C15:0 and Summed Feature 3 (C16:1 ω7c and/or C16:1 ω6c) (Supplementary Table S1). The major isoprenoid quinone was MK-7. The polar lipids were phosphatidylethanolamine, an unidentified phospholipid, an unidentified glycolipid, two unidentified aminolipids, two unidentified lipids (Fig. 3).

Figure 3
figure 3

The fatty acid profiles of strain SCS 3–6 and its relatives. (A) E. marina SCS 3–6; (B) E. shivajiensis JCM 17847T; (C) E. sediminis KCTC 52495T. PE phosphatidylethanolamine, PL unidentified phospholipid, GL unidentified glycolipid, AL unidentified aminolipid, L unidentified lipid, APL unidentified aminophospholipid.

Full size image

General genome features and genetic relatedness

The complete genome of strain SCS 3–6 using the circus program44 to visualize contained a single circular chromosome of 5,693,670 bp with a guanine-cytosine (GC) content of 40.11 mol% (Fig. 4). No plasmids were present in the strain SCS 3–6 genome. In total, 5053 coding sequence regions, 41 tRNA genes, and 5 sets of rRNA genes (5S,16S, and 23S rRNA genes) were respectively predicted (Supplementary Table S2). The GC content of strain SCS 3–6 was the lowest among all strains of current Echinicola strains.

Figure 4
figure 4

Circular genome map of Echinicola marina SCS 3–6. The circular map was generated using Circos and contain seven circles (http://www.circos.ca). Marked information is displayed from the outer circle to innermost, as follows: genomes size, CDSs on the forward stand, CDSs on the reverse stand, the distribution of rRNAs and tRNAs, GC content, GC skew.

Full size image

ANIb, ANIm, dDDH, and OrthoANI values were calculated to identify the genomic similarities of strain SCS 3–6 to the six strains of Echinicola species with available genome sequences. ANIb, ANIm, and dDDH values are presented in Table 2 and OrthoANI values are shown in Supplementary Fig. S1. The ANIb between SCS 3–6 and Echinicola pacifica DSM 19836T was 70.77 (70.66), and that between SCS 3–6 and Echinicola arenose CAU 1574T was 74.56 (74.71), substantially less than the 95% to 96% threshold for species demarcation proposed previously45. The values for ANIm among the strain SCS 3–6 and other Echinicola species were from 82.72% to 88.88%. Using dDDH analysis, strain SCS 3–6 exhibited values ranging from 18.0% with Echinicola pacifica DSM 19836T to 23.4% with Echinicola rosea JL3085T and Echinicola vietnamensis DSM 17526T (Table 2). A phylogenetic tree of seven Echinicola species based on the OrthoANI algorithm showed the analyzed Echinicola species could be divided into three clades (Supplementary Fig. S1). Strain SCS 3–6 formed a clade with Echinicola aremosa CAU 1574T. And the strain SCS 3–6 showed OrthoANI ranging from 71.41% with Echinicola pacifica DSM 19836T to 75.20% with Echinicola aremosa CAU 1574T. These values are lower than the 70% threshold for dDDH and the 95–96% threshold for ANI used for delineating prokaryotic species, thus confirming that this strain represents a new species.

Table 2 ANIb, ANIm, and dDDH values between pairs of type strains of Echinicola species.
Full size table

Core genome and pangenome of Echinicola

The genome of strain SCS 3–6 was compared to the available genomes of other Echinicola strains. The core genome sequences of individual strain were calculated. The resulting phylogenetic analysis indicated that strain SCS 3–6 formed a monophyletic clade (Fig. 5A). The pangenome of the 7 Echinicola strains possessed 10,145 gene families, while the core genome possessed 2281 gene families accounting for only 24.7% of the pangenome. The number of unique proteins in strain SCS 3–6 was 1216; specific unique protein in other Echinicola varied from 479 (E. vietnamensis) to 909 (E. pacifica) (Fig. 5B). Core and pangenome analyses of the 7 Echinicola genomes reveal an “open” pangenome fitted into a power-law regression function [f(X) = 4271.87X0.44], while the core genome was fitted into an exponential regression [f(X) = 4072.57e−0.11X] (Fig. 5C). The open pangenomes suggested that species have undergone considerable gene exchanging to extend their functional profiles46.

Figure 5
figure 5

Pan-genome analysis of Echinicola marina strains. (A) Neighbor-joining tree derived from 2281 core orthologous proteins. The tree showing the phylogenetic position of strain SCS 3–6 and closely related type strains (left) and the distribution of core genes, accessory genes and unique genes of above the strains (right). (B) Visualization of core and accessory genomes of Echinicola. (C) Mathematical modeling of the pangenome and core genome of Echinicola. (D) Bar chart showing functional proportions (based on COG categories) of different parts of the Echinicola pangenome (i.e., core, accessory, unique). The extrapolations of the Echinicola pangenome/core genome were generated using BPGA pipeline (BPGA: http://www.iicb.res.in/bpga/index.html).

Full size image

Functional COG annotation revealed that the core genome had a higher proportion of genes classified in COG categories J (translation, ribosomal structure, and biogenesis), E (amino acid transport and metabolism), F (nucleotide transport and metabolism), H (coenzyme transport and metabolism), and I (lipid transport and metabolism), which all were associated with basic biological functions and sustained life activities. The accessory genome and strain-specific genes were biased toward COG categories T (signal transduction mechanisms), K (transcription), and P (inorganic ion transport and metabolism) (Fig. 5D), which all were about informatics metabolism and were probably related to the adaption of Echinicola to various extreme habitats such as saline environments to accommodate their lifestyles (Supplementary File).

Polysaccharide utilization in the genome of E. marina

Marine Bacteroidetes are well known for their functional specialization on the decomposotion of polysaccharides which results from a great number of carbohydrate-active enzymes47. Therefore, marine bacteria may provide the most common CAZymes resources for polysaccharides degradation. The genome of SCS 3–6 harbors 299 CAZymes (Fig. 6A), including Glycoside Hydrolase (GH), Glycosyl Transferase (GT), Carbohydrate Esterase (CE), Auxiliary Activity (AA), Carbohydrate-Binding Module (CBM), and Polysaccharide Lyase (PL). The largest family found in the SCS 3–6 genome was the GH, which encoded 155 genes (Fig. 6A). The putative genes encoding GHs belonged to 43 different families with gene numbers ranging between 1 and 26, in which GH43 genes was highest. Generally, GH enzymes have great potential to hydrolyze complex carbohydrates and they are considered the key enzymes involved in carbohydrate metabolism. And GHs can degrade the most abundant biomasses. A deeper analysis toward differentiation of GHs revealed SCS 3–6 can degrade xylan. GH43 are classified based on their mode of action and substrate preference into xylanases, xylosidases, arabinofuranosidase, arabinosidase and others in SCS 3–6 genome, indicating that SCS 3–6 could have the capacity of xylan utilization. In addition, numerous genes assigned to other GH families involved in the degradation of xylan were detected including three xylanases from GH10, one xylanase from GH30, two xylosidases from GH31 and two arabinofuranosidases from GH51. Additionally, two GH115 genes predicted in SCS 3–6 can cleave glucuronic acid side chains from native xylans.

Figure 6
figure 6

Polysaccharide utilization prediction of Echinicola marina strain. (A) CAZymes distribution in SCS 3–6 genome. Different colors represent different classes of CAZymes found in the genome. The representation from the inner to outer rings are, CAZyme classes, CAZyme families, and the number of genes identified in each family, respectively. (B) Genetic organization of the predicted xylan PUL in E. marina SCS 3–6.

Full size image

PULs were manually detected based on the presence of CAZyme clusters. Some xylanolytic enzymes of E. marina SCS 3–6 were located on the multi-gene polysaccharide utilization loci (PUL), including genes that encode xynA-encoding xylanases (GH10), beta-xylosidase (GH43), alpha-glucuronidase (GH115) and one carbohydrate-binding modules (CBM6) (Fig. 6B). The xylan PUL contained genes the encode a susC–susD system and was similar to that in E. rosea JT3085T22. Xylans are heteropolymers containing xylose, arabinose, glucuronic acid, galactose and other residues. Therefore, the xylan PUL included abfA-encoding alpha-N-arabinofurnosidases (GH43) and afcA-encoding alpha-l-fucosidase (GH65). And some auxiliary activity enzymes (two esterase genes and one gene-encoding sialate O-acetylesterase) were involved in xylan degradation.

Carotenoids biosynthesis capability of E. marina

Through genome annotation, a gene cluster was shown to be directly involved in the synthesis of carotenoids. Based on these genes, crtW, crtY, crtB, crtI and crtZ (Fig. 7C), we speculated on the synthetic pathway of carotenoid production of E. marina SCS 3–6 and the production may be astaxanthin (Fig. 7A). Geranylgeranyl diphosphate (GGPP) is the direct precursor for carotenoid biosynthesis48. Phytoene is the first carotenoid formed in the bacterial carotenoid biosynthesis pathway and it is formed from the condensation of two molecules of GGPP49 by phytoene synthase (CrtB). Second, the four desaturation steps from phytoene to lycopene were mediated by a single enzyme, CrtI50. Following synthesis of lycopene, a large variety of carotenoids were produced by different lycopene cyclase processes. We found a gene of the strain genome was annotated as lycopene cyclase (CrtY). Thus, lycopene was converted to β-carotene by lycopene cyclase. The final synthesis of astaxanthin from β-carotene is a metabolic web containing several branches, according to the different participation steps and orders of β-carotene ketolase (CrtW) and β-carotene hydroxylase (CrtZ)51. Therefore, based on the presence of the crt gene in the genome, we speculated that the synthetic carotenoid may be astaxanthin. Further, the standard of astaxanthin and carotenoids extracted from the strain SCS 3–6 were analyzed by high-pressure liquid chromatography (HPLC), and the results supported the speculation (Fig. 7B).

Figure 7
figure 7

Carotenoids Biosynthesis analysis of Echinicola marina strain. (A) Proposed pathway of carotenoid biosynthesis in E. marina SCS 3–6. (B) High-pressure liquid chromatography spectra of carotenoids extracted from strain SCS 3–6 (a) and the standard of astaxanthin (b). UV detection was performed at 478 nm. (C) Carotenoid biosynthesis genes in the E. marina compared to similar gene clusters from other bacteria.

Full size image

Echinicola strains are usually orange, pink or yellow due to the presence of carotenoids pigments. In E. marina SCS 3–6 the pink pigment is cell bound and was extracted using organic solvents. To investigate the evolution of crt gene cluster, we predicted and performed a comparison of the carotenoid synthetic clusters in SCS 3–6 with other related strains (Fig. 7C). Carotenoid biosynthesis gene clusters have been found in Echinicola strains. In the genome of E. marina SCS 3–6, the cluster contained five carotenogenic genes (crtW, crtZ, crtY, crtB, and crtI) with the same orientation, one stress-responsive sigma factor gene (rpoE), one gene (merR) encoding transcriptional regulator, one gene (ispH) encoding an enzyme in the methyl erythritol phosphate pathway of isoprenoid synthesis, and one gene (oma87) encoding an outer membrane protein. Based on the comparative carotenoid synthetic gene cluster analysis, five genes encoding carotenogenic enzymes were in single gene cluster of E. marina, but the carotenoid biosynthesis gene clusters of five other available Echinicola strains contained two gene clusters that four carotenogenic genes (crtW, crtY, crtB and crtI) were found in one gene cluster and crtZ gene encoding β-carotene hydroxylase was in the other gene cluster. In E. pacifica, the crtZ gene is essentially required for astaxanthin synthesis was located in opposite orientation. The organization of rpoE–crtI–crtB–crtY–ispH–crtW–oma87 was identified and conserved in Echinicola. Of the E. marina enzymes, β-carotene hydroxylase (CrtZ) has 67%, 65%, 68%, 66% and 63% amino acid identities from E. strogylocentroti, E. soli, E. rosea, E. vietnamensis and E. pacifica, respectively. And β-carotene ketolase (CrtW) shows 72%, 71%, 73%, 67% and 67% amino acid identities from E. strogylocentroti, E. soli, E. rosea, E. vietnamensis and E. pacifica, respectively. Carotenoid accumulation in genus Echinicola stabilizes cell membranes to enable survival in various hypersaline environments. From the hypothesis of evolution and function analysis, gene deletion and genomic rearrangement caused the formation of one carotenoid synthesis gene cluster in E. marina for regulation of crt gene cluster and astaxanthin production.

Conclusion

Based on the results of phenotypic and genotypic analyses, strain SCS 3–6 is a novel species belongs to the genus Echinicola. SCS 3–6 was distinguished from closely related Echinicola species according to the phylogenetic tree constructed using 16S rRNA gene sequences and core genome sequences. We firstly research the relationships between members of the genus Echinicola. Analysis of the complete genome of SCS 3–6 indicated its polysaccharides degradation ability and carotenoid production ability.

Description of Echinicola marina sp. nov.

Echinicola marina (ma.ri’na L. fem. adj. marina, of the sea, marine).

Echinicola marina SCS 3–6 is facultative anaerobes, Gram-stain negative, non-motile by gliding. Cells are rod-shaped, and they have a width between 0.2 and 0.3 μm and a length between 1.5 and 2.0 μm. Colonies of this strain were circular, convex and pink pigmented after 2–3 days of incubation 30 °C on marine agar. Strain SCS 3–6 has exhibited positive catalase, oxidase and the hydrolysis of gelatin and casein, but negative for starch and tween 80. Strain SCS 3–6 can use various carbon sources: d-maltose, d-trehalose, d-cellobiose, gentiobiose, sucrose, d-turanose, α-d-lactose, d-melibiose, d-salicin, N-acetyl neuraminic, α-d-glucose, d-mannose, d-fructose and d-galactose.

The major cellular fatty acids are iso-C15:0 and Summed Feature 3 (C16:1 ω7c and/or C16:1 ω6c). The GenBank accession number for the 16S rRNA gene sequence of the type strain SCS 3-6T is MZ567047. The genome sequence of the type strain SCS 3-6T has been deposited in GenBank/EMBL/DDBJ under the accession number CP080025. The genome of Echinicola marina is 5,693,670 bp long and exhibits a G + C% content 40.11 mol%. The type strain is SCS 3-6T (= GDMCC 1.2220T = JCM 34403T).