Introduction

The genus Loktanella was first described by Van Trappen et al. [16] with three species, Loktanella salsilacus LMG 21507T (type strain), Loktanella fryxellensis LMG 22007T (type strain) and Loktanella vestfoldensis DSM 16212T. Many other bacterial species have been reported as belonging to this genus and at the time of writing, the genus Loktanella comprises more than 18 species (http://www.bacterio.net/deinococcus.html). Many member of the genus Loktanella have been found within different ecological niches, but mostly seawater and sea sand. A taxonomic study was performed on 2 isolated strains from a fish cage in Tongyeong, South Korea. Strains C5T and C9 identified as a member of a novel species of the genus Loktanella.

Materials and Methods

Isolation of Bacteria Strains and Culture Conditions

Two strains (designated as C5T and C9) were collected in South of Korea and isolated from a fish cage, Tongyeong (GPS: 34°59′38.19″N, 128°40′26.38″E). Two gram-negative, non-motile, strictly aerobic, short-rod shaped bacterial strains designated C5T and C9 was identified as the novel member of the genus Loktanella. To isolate the novel strains, the filtered sample were placed in 100 ml flask, inoculated with 10 ml of 10−1 diluted marine broth (Difco) and incubated aerobically in the shaking incubator (150 rpm, 28 °C) for 72 h. After incubation, using 10−5, 10−6 diluted sample, 100 µl of the aliquot was spread onto marine agar (10−1 dilution) and incubated at 28 °C for 3 days. Single colonies were isolated and aerobically sub-cultured into new marine agar and incubated again for 3 additional days. All experiments regarding strains C5T and C9 and its reference strains were stored and sub-cultured on marine agar media at pH 7.2 ± 0.2 aerobically. Type strains used as references were Loktanella salsilacus JCM 21636T [16], Loktanella atrilutea JCM 23210T [18], Loktanella fryxellensis JCM 21635T [16], Loktanella litorea KCTC 23883T [7] and Loktanella cinnabarina JCM 18161T [22] for comparative purposes.

Phylogenetic Analysis

Strains C5T and C9 were sent for 16S rRNA sequencing on an Applied Biosystems model 3730XL automated DNA-sequencing system using 9F and 1492R primers (Applied Biosystems, USA) at Macrogen Inc. Seoul, South of Korea. The complete 16S rRNA gene sequences were compiled with SeqMan software (DNASTAR Inc.) [5, 21]. The selected colonies were tentatively identified using partial 16S rRNA gene sequences, using the EzTaxon-e server (http://eztaxon-e.ezbiocloud.net) [8]. Evolutionary differences were calculated using the Kimura two-parameter model. Phylogenetic trees were constructed using the neighbour-joining [14], maximum-parsimony [7] and maximum-likelihood methods in MEGA5 program [20] with bootstrap values based on 1000 replications [3, 4].

Phenotypic and Biochemical Characteristics

Cells were grown on marine agar for 2–3 days before their descriptive examination. Gram reaction was carried out the classic Gram staining described by Doetsch [1]. API ZYM, 20NE, and ID32GN were used according to the manufacturer recommendations (bioMérieux) in order to examine their utilization of carbon source and enzymatic activities. p-dimethyl aminobenzaldehyde with HCl and amyl alcohol (Kovac`s reagent) in SIM agar was used for indole test. For methyl red test and Voges-Proskauer test, incubated MR-VP broth for 2 days with methyl red indicator and Barritt`s reagents. Citrate utilization test was checked Simmons citrate agar. Phenylalanine deaminase test was assessed using 10 % (w/v) ferric chloride solution. Nitrate reduction test was basically a nutrient broth supplemented with 0.1 % potassium nitrate as the nitrate substrate. C5T and C9 ability to reduce nitrates to nitrites was determined by the addition of sulfanilic acid and α-naphtylamine. Casein test was conducted on marine agar with 2 % casein acid hydrolysate. Anaerobic growth was examined in serum bottles with marine broth and the upper air layer was replaced with nitrogen. Range and optimal temperature growth on marine broth was examined for 3 days at different combinations of temperatures (4, 10, 15, 20, 25, 30, 32, 37, 40 and 45 °C), and (4.0, 5.0, 6.0, 7.0, 8.0 and pH 9.0) at 30 °C. Optimal salt requirement on marine agar was examined for 3 days at levels 0, 1, 2, 3, 5, 7, 10, 15, 20 and 25 % NaCl (w/v). Growth media was observed on only marine agar.

Chemotaxonomic Characteristics

Respiratory quinones were extracted from freeze-dried bacterial cells of strains C5T and C9 with chloroform/methanol (2:1, v/v), evaporated under vacuum and re-extracted in hexane. The quinone solution was purified using silica Sep-Pak Vac 6 cc cartridges (Waters Solid Phase Extraction) and subsequently analyzed by high performance liquid chromatography (HPLC), as described previously [6].

The analysis of fatty acid methyl ester was performed after growth of bacteria on marine agar for 3 days at 30 °C and exponential phase bacterial cells were harvested in order to prepare, separate, and identify the fatty acid methyl esters with the Sherlock Microbial Identification System, produced by MIDI, Inc., Newark, DE, USA [15].

For detection of polar lipids, cells of strains C5T and C9 were grown in marine broth at 30 °C, examined by two-dimensional TLC [9, 12, and 13] and identified by two-dimensional TLC followed by spraying with appropriate detection reagents. Total lipid profile was detected by spraying with molybdophosphoric acid solution (Sigma-Aldrich; St. Louis, MO) followed by heating at 150 °C; free-aminolipids by spraying with 0.2 % (w/v) ninhydrin solution followed by heating at 100 °C for 5 min; phospholipids by spraying with Zinzadze reagent (Sigma-Aldrich; St. Louis, Mo).

Genomic Characteristics

In order to determine the DNA G + C content of strains C5T and C9, genomic DNA was extracted using the Marmur method [10]. DNA was separated by nuclease P1 (100 U/ml, pH 5.3) at 37 °C for 1 h. The separated nucleosides were then analyzed using reverse-phase HPLC [11, 19]. DNA–DNA hybridizations were carried out with photobiotin-labelled probes in microplate wells as described by Ezaki et al. [2] using a Multilabel Reader (Perkin Elmer) for fluorescence measurements.

Results and Discussion

Morphological and Physiological Characteristics

Strains C5T and C9 were found to be Gram negative, strictly aerobic, short rod shaped, 1.0–3.0 µm long and 0.5–1.0 µm wide. When cultured on marine agar at 30 °C, colonies were beige color, circular, and convex. Strains C5T and C9 grew between 25–32 °C and pH 7.0–9.0. Optimum growth of this novel strains were observed at 30 °C and pH 8.0. It also grew in the presence of 0–10 % NaCl (w/v). Optimum NaCl concentration was 2 % (w/v). Results for the physiological characteristics of Strains C5T and C9 are summarized in the species description and comparison of discriminating characteristics of reference strains are shown in Table 1.

Table 1 Comparison of phenotypic characters of C5T, C9 and the type strains of related Loktanella species
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Phylogenetic Analysis

Almost complete sequences of the 16S rRNA genes were compiled with SeqMan software (DNASTAR Inc.). The 16S rRNA gene sequence of strains C5T and C9 (GenBank/EMBL/DDBJ accession numbers KP716799T and KP883300, respectively) show 99.2 % similarity. A phylogenetic analysis of the 16S rRNA gene sequences showed that strains (C5T and C9) belongs to the phylum Proteobacteria, class Alphaproteobacteria, order Rhodobacterales and family Rhodobacteraceae. Loktanella salsilacus LMG 21507T (96.8 %), Loktanella atrilutea IG8T (96.2 %), Loktanella fryxellensis LMG 22007T (96.0 %), Loktanella litorea DPG-5T (95.4 %), Loktanella cinnabarina LL-001T (95.1 %) were determined to be the most closely related neighbours. The strains C5T and C9 joined the cluser comprissed of the member of the genus Loktanella by neighbor-joining tree (Fig. 1) with the high bootrap (89 %) value. The phylogenic position of the trains are also cofnrimed by maximum likelihood and maximum parsimony phylogenetic trees (See supplementary figures S1 and S2).

Fig. 1
figure 1

Phylogenetic tree of strains C5T and C9 with closely related Loktanella type strains bas ed on 16S rRNA gene sequence comparisons. Neighbour-joining phylogenetic tree based on nearly complete 16S rRNA gene sequences showing the relationship between strain C5T and the type strains of closely related members of the genus Loktanella. Bootstrap values of >50 % (percentages of 1000 replications) are shown at branching points. The sequences used for the comparative study are included in parentheses

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Chemotaxonomic Characteristics

The cellular fatty acids of strains C5T and C9 were C18:1 ω7c (>77 %) and C16:0(>10 %). Minor amounts (<10 %) were found for C10:0 3OH, C14:0, C14:0 3OH/C16:1 iso I, C16:1 ω7c/C16:1 ω6c (Summed Feature 3), C17:1 ω8c, C17:0, C18:0, C18:1 ω7c 11-methyl, Un18.846/C19:1 ω6c (Summed Feature 7), C19:0 10 methyl. The fatty acid profile of strains C5T and C9 are shown in Table 2 and compared with other Loktanella species.

Table 2 Fatty acid composition of strain C5T, C9 and the type strains of closely related species in the genus Loktanella. Taxa: 1, Strain C5T; 2. Strain C9; 3, L. salsilacus JCM 21636T; 4, L. atrilutea JCM 23210T; 5, L. fryxellensis JCM 21635T; 6, L. litorea KCTC 23883T; 7. L. cinnabarina JCM 18161T
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Polar lipid profile contained phosphatidylglycerol (PG), phosphatidycholine (PC), diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), unidentified amino lipid (AL) and unidentified lipids (L1–3). Strains C5T and C9 are compared polar lipid in Fig. 2 but strain C9 have unidentified lipid more than C5T. Loktanella species are characterized as possessed Q-10 as the sole respiratory quinone. Strain C5T and C9 also possessed Q-10 as the respiratory quinone.

Fig. 2
figure 2

Result of polarlipid picture C5T (a) and C9 (b)

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Genomic characteristics

The genomic DNA G + C content of strains C5T and C9 was 61.0 and 61.6 mol%. Both the strains exhibited high levels of DNA–DNA relatedness values (>72 %), reciprocally, indicating them to belong to the same species (Table 3) [17].

Table 3 DNA–DNA hybridization of strain C5T and C9 with L. salsilacus JCM 21636T
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Taxonomic Conclusion

To conclude, the morphological, physiological, chemotaxonomic and phylogenetic data obtained during this study clearly showed that strains C5T and C9 belong to the genus Loktanella. Assimilation of l-arabinose, citrate, gluconate, d-mannitol, indole can easily distinguish these strains from phylogenetically close species. We propose strain C5T as the novel species of the genus Loktanella, with the name Loktanella aquimaris sp. nov.

Description of Loktanella aquimaris sp. nov

Loktanella aquimaris (a.qui.máris. L. n. aqua, water; L. gen. n. maris, of the sea; N.L. gen. n. aquimaris, of the water of the sea).

Gram negative, strictly aerobic, non-motile, short rod shaped, 1.0–3.0 µm long and 0.5–1.0 µm wide. Colonies were circular, convex and beige on marine agar. Growth of strain C5T and C9 occurred grew between temperatures 25–32 °C (optimum 30 °C). The pH range is 7.0–9.0 (optimum pH 8.0). Growth on salt level 0–10 % NaCl (w/v) (optimum 2–3 %). Casein, citrate, H2S, nitrate and nitrite reduction, carbonate fermentation, gelatin hydrolysis tests was negative but indole, methyl red, phenylalanine deaminase tests was positive. Positive for alkaline phosphatase, acid phosphatase, naphtol-AS-BI-phosphohydrolase, N-acetyl-β-glucosaminidase, Esterase (C4), esterase (C8), β-glucosidase (esculin hydrolysis), β-galactosidase (PNPG), d-glucose, glycogen, l-histidine, indole, leucine arylamidase, suberate, l-alanine and valine arylamidase as a sole carbon and energy source. Variable results are obtained for adipate, N-acetyl-d-glucosamine, cystine arylamidase, l-fucose, gluconate, d-glucose, 3-hydroxybenzoate, 4-hydroxybenzoate, 2-ketogluconate (α), d,l-lactate, lipase (C14), d-mannose, d-mannitol, d-maltose, d-melibiose, urease and n-valerate. Acetate, l-arabinose, arginine dihydrolase, caprate, α-chymotrypsin, citrate, α-fucosidase, α-galactosidase, β-glucuronidase, β-galactosidase (ONPG), α-glucosidase (starch hydrolysis), d,l-3-hydroxybutyrate, myo-Inositol, 5-ketogluconate, α-mannosidase, phenyl acetate, protease, l-proline, propionate, d-ribose, salicin, d-sorbitol and trypsin was not utilized as a sole carbon source and enzyme. All data were obtained in this study. +, positive; −, negative; w, weak (Table 1).

Major polar lipids were phosphatidylglycerol (PG), phosphatidycholine (PC), diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), unidentified amino lipid (AL) and minor polar lipids were unidentified lipid (Fig. 2). The predominant isoprenoid quinone detected in strain was ubiquinone-10 (Q-10). Major fatty acids and isoprenoid quinone of the type strain were C18:1 ω7c and C16:0 and ubiquinone-10 respectively. The DNA G + C content was 61.0–61.4 mol%. The type strains C5T (=KEMB3-892T = JCM 30382T) and C9 (=KEMB 3-893 = JCM 30383T) was isolated from seawater in Tongyeong, South of Korea.