Antonie van Leeuwenhoek (2014) 106:449–455 DOI 10.1007/s10482-014-0213-5
ORIGINAL PAPER
Marinomonas profundimaris sp. nov., isolated from deep-sea sediment sample of the Arctic Ocean Xiuhua Bai • Qiliang Lai • Chunming Dong Fuying Li • Zongze Shao
•
Received: 22 January 2014 / Accepted: 9 June 2014 / Published online: 22 June 2014 Ó Springer International Publishing Switzerland 2014
Abstract A taxonomic study was carried out on strain D104T, which was isolated from deep-sea subsurface sediment sample from the Arctic Ocean. The bacterium was found to be Gram-negative, oxidase negative and catalase weakly positive, rod shaped, motile by means of polar flagellum. The organism grows between 4 and 37 °C (optimum 25–28 °C) and 0.5–6 % NaCl (optimum 3 %). Phylogenetic analysis based on 16S rRNA gene sequences
Xiuhua Bai and Qiliang Lai have contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s10482-014-0213-5) contains supplementary material, which is available to authorized users. X. Bai Q. Lai C. Dong F. Li Z. Shao State Key Laboratory Breeding Base of Marine Genetic Resources, Xiamen, China X. Bai Q. Lai C. Dong F. Li Z. Shao (&) Key Laboratory of Marine Genetic Resources, Third Institute of Oceanography, SOA, Xiamen, China e-mail: [email protected]
indicated that strain D104T belongs to the genus Marinomonas, with highest sequence similarities of 97.7 % to Marinomonas ushuaiensis DSM 15871T, followed by M. dokdonensis DSW10-10T (96.9 %), M. arenicola KMM 3893T (96.7 %), M. arctica 328T (96.6 %) and other 18 species of the genus Marinomonas (94.4–96.5 %). The average nucleotide identity and estimated DNA–DNA hybridization values between strain D104T and M. ushuaiensis DSM 15871T were 84.24 % and 20.80 ± 2.33 % respectively. The principal fatty acids were C16:0, sum in feature 3 (C16:1x7c/C16:1x6c), sum in feature 8 (C18:1x7c/C18:1x6c) and C12:1 3OH. The G ? C content of the chromosomal DNA was determined to be 44.8 mol%. The respiratory quinone was found to be Q8 (100 %). Polar lipids include phosphatidylglycerol and phosphatidylethanolamine as major phospholipids and aminolipid and phospholipid as minor components. The results of the genotypic and phenotypic analyses indicate that strain D104T represents a novel species of the genus Marinomonas, for which the name Marinomonas profundimaris sp. nov. is proposed, with the type strain D104T (=MCCC 1A07573T = LMG 27696T).
X. Bai Q. Lai C. Dong F. Li Z. Shao Collaborative Innovation Center of Deep Sea Biology, Xiamen, China
Keywords Marinomonas profundimaris sp. nov. Arctic Ocean Deep sea sediment
X. Bai Q. Lai C. Dong F. Li Z. Shao Key Laboratory of Marine Genetic Resources of Fujian Province, Xiamen 361005, China
Abbreviation MCCC Marine Culture Collection of China
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Introduction The genus Marinomonas was proposed by Van Landschoot and De Ley (Van Landschoot and De Ley 1983) and currently comprises 22 recognized species (http://www.bacterio.cict.fr/m/marinomonas. html). The members of the genus Marinomonas are Gram-negative, helical, curved or straight rods, motile by means of polar flagellum, aerobic (Espinosa et al. 2010). The strains of the genus Marinomonas were isolated from different marine environments, such as Pacific Ocean (Baumann et al. 1972), Black Sea (Ivanova et al. 2005), Mediterranean Sea (Solano and Sanchez-Amat 1999), Arctic Ocean (Zhang et al. 2008). This paper focuses on taxonomic study of strain D104T which was isolated during our investigation of the diversity of polycyclic aromatic hydrocarbons (PAHs)-degrading bacteria of the deep-sea sediments of the Arctic Ocean. Comparative 16S rRNA gene sequence analysis indicated that strain D104T belongs to the genus Marinomonas. The draft genome sequence of strain had been reported including 62 contigs with genome size of approximately 3.83 Mbp (Dong et al. 2014). The aim of this work was to determine the exact taxonomic position of strain D104T.
Materials and methods
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phenotypic tests were carried out on marine agar 2216 (MA, BD) plates unless noted otherwise. Two type strains, Marinomonas ushuaiensis DSM 15871T and Marinomonas communis DSM 5604T, were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Germany) and used as reference strains. All strains were stored at -80 °C in marine broth 2,216 (BD) supplemented with 16 % (v/v) glycerol for maintenance. Phenotypic characterization Gram reaction, catalase and oxidase activities, hydrolysis of aesculin, Tween 80, casein and starch, the growth range of temperature and pH, tolerance to NaCl and cell morphology were studied as previously described (Lai et al. 2009). Antibiotic susceptibility tests were performed using the disc diffusion method as described elsewhere (Shieh et al. 2003). Anaerobic growth was examined on marine agar 2,216 supplemented with nitrate (1 g/L) incubated in a jar with the Anoxomat Mark II Anaerobic System (Mart Microbiology, Holland). API 20NE, API 20E, API ZYM strips (bioMe´rieux) and Biolog GN2 were carried out according to the manufacturer’s instructions, with the single modification of adjusting the NaCl concentration to 3.0 % in all tests. M. ushuaiensis DSM 15871T and M. communis DSM 5604T were tested at the same time.
Bacterial strains isolation and cultivation Phylogenetic analysis of 16S rRNA gene sequence The deep-sea sediments were sampled using a box sampler (EKG-M, BEIJING PURITY INSTRUMENT CO., LTD.) during the fourth Chinese National Arctic Research Expedition of the ‘‘XUELONG’’ icebreaker in July 2010. The sampling site BN12 was located at Makarov basin (W170°290 , N87°040 ) in the Arctic Ocean, with the depth of 4,000 m. The sediment core was firstly sampled by a box sampler, then subsampled by a push core sampler prior to releasing the box corer on deck. Subsequently, the core was sliced into layers at depth intervals of 2 cm, and then about 5 g sediment of each layer was used to set up PAH-degrading consortia. The strain D104T was isolated from the consortium of subsurface sediment layer (8–12 cm) on 216L medium as described elsewhere (Lai et al. 2009). The plates were incubated at 25 °C 2 weeks. The routine cultivation of the strain D104T and most
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The 16S rRNA gene sequence of strain D104T was determined from the draft genome (Dong et al. 2014). The 16S rRNA gene was also amplified by PCR using primers described previously (Liu and Shao 2005). Sequence similarity of 16S rRNA gene was determined using the EzTaxon-e server (Kim et al. 2012). Sequences of 23 strains of related taxa were obtained from the GenBank database. Phylogenetic analysis was performed using MEGA version 5.0 (Tamura et al. 2011). Distances (distance options according to the Kimura two-parameter model) and clustering according to the neighbour-joining (NJ) method of Saitou and Nei (1987), maximum likelihood (ML) (Felsenstein 1981) and minimum evolution (ME) method of Rzhetsky and Nei (1993) were determined by using bootstrap values based on 1,000 replications.
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Genome sequencing, G ? C content, average nucleotide identity (ANI) and DNA–DNA hybridization (DDH) estimation The genome of M. ushuaiensis DSM 15871T was sequenced at Shanghai Majorbio Bio-pharm Technology Co., Ltd (Shanghai, China) using Solexa pairedend (500 bp library) sequencing technology. A total of 500 Mbp clean data for M. ushuaiensis DSM 15871T were generated to reach about 100-fold depth of coverage using an Illumina/Solexa Genome Analyzer IIx (Illumina, SanDiego, CA). The genome was assembled using SOAPdenovo2 (Luo et al. 2012). The accession number of M. ushuaiensis DSM 15871T is JAMB00000000. The draft genome sequence of the strain D104T (AYOZ00000000) was obtained from NCBI (Dong et al. 2014). The G ? C content of the chromosomal DNA was calculated on the basis of its whole genome sequence. The ANI was calculated using J-specises (V1.2.1) as described elsewhere (Richter and Rossello-Mora 2009). DNA–DNA hybridization (DDH) value was estimated using the genome-to-genome distance calculator (GGDC2.0) (Auch et al. 2010a, b; Meier-Kolthoff et al. 2013).
451 Table 1 Physiological characteristics of Marinomonas profundimaris D104T and related species of the genus Marinomonas Characteristic
1
2
3
Oxidase
–
–
?
Growth at: 4 °C
Strain D104T was found to be Gram-negative, aerobic, motile by means of a single flagellum and rod-shaped with the size of 0.6–0.7 lm width and 1.5–1.6 lm length (Supplementary Fig S1). Colonies on marine agar
–
–
?
40 °C
–
–
?
44.8a
43.6 (41.1a)
46.3
API 20NE Arginine dihydrolase
w
?
-
ß-galactosidase
w
-
?
ß-glucosidase (Aesculin hydrolysis)
?
-
w
Trisodium citrate
?
-
?
Urease
–
?
-
Utilization of L-arabinose, N-acetyl-glucosamine
?
-
-
Utilization of malic acid, potassium gluconate Utilization of D-maltose
?
-
?
?
w
?
Esterase(C4), achymotrypsin, naphtol-ASBl-phosphoamidase
–
?
?
Esterase lipase (C8), trypsin
–
?
w
a-galactosidase
–
w
–
b-galactosidase
?
–
–
a-glucosidase, b-glucosidase
?
–
?
w
w
?
Acetoin production (Voges Proskauer)
-
?
-
Tryptophane deaminase
?
w
?
Oxidation arabinose
?
-
?
Oxidation inositol
?
-
w
Oxidation mannitol
?
-
-
Oxidation melibiose, saccharose, sorbitol Susceptibility to:
?
-
w
S
R
S
Enzymatic activity (API ZYM)
Cystine aminopeptidase API 20E
Ampicillin (10 lg), erythromycin (15 lg), penicillin G (10 lg)
Results and discussion Phenotypic characteristics
?
?
DNA G ? C content (mol%)
Determination of fatty acid, isoprenoid ubiquinone and polar lipid compositions The whole cells grown on marine agar 2,216 plate at 25 °C for 48 h were harvested. Fatty acids were saponified, methylated and extracted using the standard protocol of MIDI (Sherlock Microbial Identification System, version 6.0B). The fatty acids were identified by using the TSBA6.0 database of the Microbial Identification System (Sasser 1990). M. ushuaiensis DSM 15871T and M. communis DSM 5604T were tested at the same time. Analysis of respiratory quinone and polar lipid were carried out by the Identification Service of the DSMZ, Braunschweig, Germany.
?
35 °C
Strains: 1, D104T; 2, M. ushuaiensis DSM 15871T (Prabagaran et al. 2005); 3, M. communis DSM 5604T (Chimetto et al. 2011) API 20NE, API 20E, API ZYM and susceptibility to antibiotics data are performed in this study. Characteristics are scored as: ? positive, - negative, w weak, S sensitive, R resistive a
Data from draft genome sequence
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Marinomonas arctica 328T (DQ492749) Marinomonas hwangdonensis HDW-15T (JN087530) Marinomonas polaris CK13T (AJ833000) Marinomonas primoryensis KMM 3633T (AB074193) Marinomonas rhizomae IVIA-Po-145T (EU188443)
Marinomonas foliarum IVIA-Po-155T (EU188444) Marinomonas alcarazii IVIA-Po-14bT (EU188442)
77 98
Marinomonas pontica 46-16T (AY539835)
Marinomonas profundimaris D104T (KC565667) Marinomonas ushuaiensis U1T (AJ627909) 95 Marinomonas arenicola KMM 3893T (AB467281) 97 Marinomonas pollencensis IVIA-Po-185T (EU188441) Marinomonas dokdonensis DSW10-10T (DQ011526) Marinomonas posidonica IVIA-Po-181T (CP002771) 97 100 Marinomonas aquiplantarumi IVIA-Po-159T (EU188447) Marinomonas brasilensis R-40503T (GU929940) Marinomonas vaga ATCC 27119T (X67025)
63
Marinomonas communis LMG 2864T (DQ011528) Marinomonas aquimarina CECT 5080T (AJ843077)
65 95
100
Marinomonas fungiae AN44T (JQ409370) Marinomonas ostreistagni UST010306-043T (AB242868) Marinomonas mediterranea MMB-1T (CP002583) Marinomonas balearica IVIA-Po-101T (EU188448) Marinobacterium lutimaris AN9T (FJ230839)
0.01 Fig. 1 Neighbour-joining tree showing the phylogenetic position of Marinomonas profundimaris D104T within genus Marinomonas based on 16S rRNA gene sequence similarities. Filled circles indicate nodes that were also recovered in maximum-likelihood and minimum evolution trees based on
the same sequences. Bootstrap values (expressed as percentages of 1,000 replications) are shown at branch points. Bar, 0.01 nucleotide substitution rate (Knuc) units. Marinobacterium lutimaris AN9T was used as the outgroup
2,216 are round, dull white, non-pigmented, nonluminescent with irregular margins and 2–3 mm in diameter after 48 h incubation at 25 °C. Growth occurs at 4–37 °C (optimum 25–28 °C) and pH 5–10 (optimum 7) and in presence 0.5–6 % of NaCl (optimum 3 %). Strain D104T is found to be sensitive to ampicillin (10 lg/per disk, Oxoid), carbenicillin (100), cefalexin (30), cefazolin (30), chloromycetin (30), ciprofloxacin (5), erythromycin (15), furazolidone (15), furazolidone (5), gentamicin (10), kanamycin (30), minomycin (30), neomycin (10), norfloxacin (10), ofloxacin (5), penicillin G (10), polymyxin B (30 IU), streptomycin (10),
tetracycline (30), vibramycin (30); resistant to clindamycin (2), lincomycin (2), metronidazole (5), oxacillin (1), piperacillin (100) and vanoomycin (30). Other characteristics are given in species description. The characteristic phenotypic features of strain D104T and two reference type strains are shown in Table 1.
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Phylogenetic analysis based on 16S rRNA gene sequence similarities A nearly full-length 16S rRNA gene sequence (1,503 nt of strain D104T was used in phylogenetic
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analysis. As shown in Fig. 1, phylogenetic tree based on 16S rRNA gene sequence similarities showed that strain D104T formed a robust clade with M. ushuaiensis DSM 15871T within the genus Marinomonas (95 % bootstrap support). Same topology was retrieved in maximum-likelihood and minimum evolution trees (Supplementary materials Figs. S2 and S3). Strain D104T was found to have the highest sequence similarity of 97.7 % to M. ushuaiensis DSM 15871T, followed by M. dokdonensis DSW10-10T (96.9 %), M. arenicola KMM 3893T (96.7 %), M. arctica 328T (96.6 %) and other 18 species of the genus Marinomonas (94.4–96.5 %). DNA G ? C content, ANI and DDH The DNA G ? C content of strain D104T was found to be 44.8 mol% which is in the range of the genus Marinomonas (i.e., 41–50 mol%) (Espinosa et al. 2010). The DNA G ? C content of M. ushuaiensis DSM 15871T was estimated to be 41.3 mol% according to its draft genome sequence, which was comparable to previously reported value of 43.6 mol%. The ANI of strain D104T and M. ushuaiensis DSM 15871T was found to be 84.24 %. This value is significantly lower than the suggested threshold range of 95–96 % (Richter and Rossello-Mora 2009). The DDH estimated value between two strains was found to be 20.80 ± 2.33 %, which is also significantly lower than the recommended threshold of 70 % (Wayne et al. 1987). These results suggested that strain D104T represents a novel genomic species of the genus Marinomonas. Chemotaxonomic characteristics The principal fatty acids of strain D104T were found to be sum in feature 3 (C16:1x7c/C16:1x6c) (27.1 %), sum in feature 8 (C18:1x7c/C18:1x6c) (39.7 %), C16:0 (12.3 %) and C12:1 3OH (5.8 %) (Table S1). The fatty acids patterns of all tested strains appeared to be similar with the same characteristics fatty acids ([10 %) and C10:0 3OH ([2 %) (Espinosa et al. 2010). The respiratory quinone of strain D104T was found to be Q-8 (100 %) in agreement with previously reported data (Yoon et al. 2005; Jung et al. 2012). Polar lipids of strain D104T were found to consist of a phosphatidylglycerol (PG), a phosphatidylethanolamine (PE), an aminolipid (AL) and a phospholipid (PL) (Supplementary Fig. S4). The major polar lipid,
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PG and PE, were also present in type species M. communis and other species of the genus Marinomonas (Gupta et al. 2006; Zhang et al. 2008; Jung et al. 2012). Taxonomic conclusion Strain D104T could be assigned to the genus Marinomonas based on the results of phylogenetic analysis, phenotypic analysis and chemotaxonomic data. However, the strain D104T could be distinguished from the close phylogenetic relatives by several phenotypic characteristics listed in Table 1. On the basis of the results presented in this study and taking into account DNA–DNA hybridization values below 70 % it is proposed that strain D104T can be classified as novel species of the genus Marinomonas for which the name Marinomonas profundimaris sp. nov. is proposed. Description of Marinomonas profundimaris sp. nov Marinomonas profundimaris (pro.fun.di.ma’ris. L. adj. profundus deep; L. gen. n. maris from/of the sea; N.L. gen. n. profundimaris from/of the deep-sea, reflecting from where the type strain was isolated). Cells are Gram-negative, rod-shaped, about 0.6–0.7 lm width and 1.5–1.6 lm length, motile with a single polar flagellum. Aerobic. Chemoorganotroph with respiratory metabolism. The colonies are round, dull white and non-pigmented with irregular margins, 2–3 nm in diameter, convex, smooth on marine agar. Catalase weakly positive, oxidase negative. Negative for Tween 80 and casein hydrolysis, positive for amylase. Requires NaCl for growth. Growth occurs in media with 0.5–6 % of NaCl (optimum 3 %). Temperature for growth ranges from 4 to 37 °C (optimum 25-28 °C). The pH for growth ranges from 5 to 10 (optimum pH 7). According to API ZYM, positive for acid phosphatase, alkaline phosphatase, leucine aminopeptidase, valine aminopeptidase, a-glucosidase, bgalactosidase, b-glucosidase and weakly positive for cystine aminopeptidase, lipase (C14); negative for bglucuronidase, esterase lipase (C8), esterase (C4), N-acetyl-b-glucosaminidase, naphtol-AS-Bl-phosphoamidase, trypsin, a-chymotrypsin, a-fucosidase, a-galactosidase, a-mannosidase. According to API 20NE, positive for arginine dihydrolase, b-galactosidase (weak), b-glucosidase (aesculin hydrolysis) and utilization of D-glucose, D-mannitol, D-mannose,
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L-arabinose, malic acid, potassium gluconate and trisodium citrate; negative for D-glucose fermentation, gelatinase, urease, indole production, nitrate reduction and utilization of capric acid, phenylacetic acid, adipic acid, D-maltose and N-acetyl-glucosamine. According to API 20E, positive for citrate, tryptophane deaminase, arginine dihydrolase (weak), ß-galactosidase (weak) and oxidation of arabinose, inositol, mannitol, melibiose, saccharose, sorbitol; negative for gelatinase, H2S production, indole production, lysine decarboxylase, ornithine decarboxylase and oxidation of amygdalin, glucose and rhamnose. According to Biolog GN2 system, utilizes bromo succinic acid, cis-aconitic acid, citric acid, D,L-lactic acid, D-arabitol, D-cellobiose, D-fructose, D-galactose, D-gluconic acid, D-mannitol, D-mannose, D-melibiose, D-sorbitol, Dtrehalose, glucose-6-phosphate, glycerol, glycyl-Lglutamic acid, inosine, L-asparagine, L-aspartic acid, L-glutamic acid, L-ornithine, L-proline, maltose, m-inositol, succinic acid, sucrose, turanose, uridine, urocanic acid, a-D-glucose, a-keto glutaric acid and c-amino butyric acid; weakly positive for 2,3-butanediol, 2-aminoethanol, acetic acid, D,L-a-glycerol phosphate, D-alanine, dextrin, D-galacturonic acid, D-glucuronic acid, D-raffinose, D-saccharic acid, glucose-1-phosphate, glycogen, glycyl-L-aspartic acid, L-alanine, L-alanyl-glycine, L-pyroglutamic acid, L-serine, malonic acid, methyl pyruvate, mono-methylsuccinate, N-acetyl-D-glucosamine, putrescine, quinic acid, succinamic acid, thymidine, tween 40, tween 80, b-hydroxy butyric acid, c-hydroxy butyric acid; negative for others. The main cellular fatty acids are sum in feature 3 (C16:1x7c/C16:1x6c), sum in feature 8 (C18:1x7c/C18:1x6c), C16:0 and C12:1 3OH. The respiratory quinone is Q-8 (100 %). Phosphatidylglycerol (PG), phosphatidylethanolamine (PE) and aminolipid (AL) are main polar lipids. The G ? C content of the chromosomal DNA of the type strain is 44.8 mol%. The type strain D104T (= MCCC 1A07573T = LMG 27696T), isolated from deep sea sediment sample from Arctic Ocean. The GenBank accession number of the genome is AYOZ00000000. Acknowledgments This work was financially supported by Public Welfare Project of SOA (201005032), COMRA program (No. DY125-15-R-01), National Natural Science Foundation of China (41206158), the China Polar Environment Investigation and Estimate Project (2012-2015) and National Infrastructure of Natural Resources for Science and Technology Program of China (No. NIMR-2013-9).
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ORIGINAL PAPER
Marinomonas profundimaris sp. nov., isolated from deep-sea sediment sample of the Arctic Ocean Xiuhua Bai • Qiliang Lai • Chunming Dong Fuying Li • Zongze Shao
•
Received: 22 January 2014 / Accepted: 9 June 2014 / Published online: 22 June 2014 Ó Springer International Publishing Switzerland 2014
Abstract A taxonomic study was carried out on strain D104T, which was isolated from deep-sea subsurface sediment sample from the Arctic Ocean. The bacterium was found to be Gram-negative, oxidase negative and catalase weakly positive, rod shaped, motile by means of polar flagellum. The organism grows between 4 and 37 °C (optimum 25–28 °C) and 0.5–6 % NaCl (optimum 3 %). Phylogenetic analysis based on 16S rRNA gene sequences
Xiuhua Bai and Qiliang Lai have contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s10482-014-0213-5) contains supplementary material, which is available to authorized users. X. Bai Q. Lai C. Dong F. Li Z. Shao State Key Laboratory Breeding Base of Marine Genetic Resources, Xiamen, China X. Bai Q. Lai C. Dong F. Li Z. Shao (&) Key Laboratory of Marine Genetic Resources, Third Institute of Oceanography, SOA, Xiamen, China e-mail: [email protected]
indicated that strain D104T belongs to the genus Marinomonas, with highest sequence similarities of 97.7 % to Marinomonas ushuaiensis DSM 15871T, followed by M. dokdonensis DSW10-10T (96.9 %), M. arenicola KMM 3893T (96.7 %), M. arctica 328T (96.6 %) and other 18 species of the genus Marinomonas (94.4–96.5 %). The average nucleotide identity and estimated DNA–DNA hybridization values between strain D104T and M. ushuaiensis DSM 15871T were 84.24 % and 20.80 ± 2.33 % respectively. The principal fatty acids were C16:0, sum in feature 3 (C16:1x7c/C16:1x6c), sum in feature 8 (C18:1x7c/C18:1x6c) and C12:1 3OH. The G ? C content of the chromosomal DNA was determined to be 44.8 mol%. The respiratory quinone was found to be Q8 (100 %). Polar lipids include phosphatidylglycerol and phosphatidylethanolamine as major phospholipids and aminolipid and phospholipid as minor components. The results of the genotypic and phenotypic analyses indicate that strain D104T represents a novel species of the genus Marinomonas, for which the name Marinomonas profundimaris sp. nov. is proposed, with the type strain D104T (=MCCC 1A07573T = LMG 27696T).
X. Bai Q. Lai C. Dong F. Li Z. Shao Collaborative Innovation Center of Deep Sea Biology, Xiamen, China
Keywords Marinomonas profundimaris sp. nov. Arctic Ocean Deep sea sediment
X. Bai Q. Lai C. Dong F. Li Z. Shao Key Laboratory of Marine Genetic Resources of Fujian Province, Xiamen 361005, China
Abbreviation MCCC Marine Culture Collection of China
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Introduction The genus Marinomonas was proposed by Van Landschoot and De Ley (Van Landschoot and De Ley 1983) and currently comprises 22 recognized species (http://www.bacterio.cict.fr/m/marinomonas. html). The members of the genus Marinomonas are Gram-negative, helical, curved or straight rods, motile by means of polar flagellum, aerobic (Espinosa et al. 2010). The strains of the genus Marinomonas were isolated from different marine environments, such as Pacific Ocean (Baumann et al. 1972), Black Sea (Ivanova et al. 2005), Mediterranean Sea (Solano and Sanchez-Amat 1999), Arctic Ocean (Zhang et al. 2008). This paper focuses on taxonomic study of strain D104T which was isolated during our investigation of the diversity of polycyclic aromatic hydrocarbons (PAHs)-degrading bacteria of the deep-sea sediments of the Arctic Ocean. Comparative 16S rRNA gene sequence analysis indicated that strain D104T belongs to the genus Marinomonas. The draft genome sequence of strain had been reported including 62 contigs with genome size of approximately 3.83 Mbp (Dong et al. 2014). The aim of this work was to determine the exact taxonomic position of strain D104T.
Materials and methods
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phenotypic tests were carried out on marine agar 2216 (MA, BD) plates unless noted otherwise. Two type strains, Marinomonas ushuaiensis DSM 15871T and Marinomonas communis DSM 5604T, were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Germany) and used as reference strains. All strains were stored at -80 °C in marine broth 2,216 (BD) supplemented with 16 % (v/v) glycerol for maintenance. Phenotypic characterization Gram reaction, catalase and oxidase activities, hydrolysis of aesculin, Tween 80, casein and starch, the growth range of temperature and pH, tolerance to NaCl and cell morphology were studied as previously described (Lai et al. 2009). Antibiotic susceptibility tests were performed using the disc diffusion method as described elsewhere (Shieh et al. 2003). Anaerobic growth was examined on marine agar 2,216 supplemented with nitrate (1 g/L) incubated in a jar with the Anoxomat Mark II Anaerobic System (Mart Microbiology, Holland). API 20NE, API 20E, API ZYM strips (bioMe´rieux) and Biolog GN2 were carried out according to the manufacturer’s instructions, with the single modification of adjusting the NaCl concentration to 3.0 % in all tests. M. ushuaiensis DSM 15871T and M. communis DSM 5604T were tested at the same time.
Bacterial strains isolation and cultivation Phylogenetic analysis of 16S rRNA gene sequence The deep-sea sediments were sampled using a box sampler (EKG-M, BEIJING PURITY INSTRUMENT CO., LTD.) during the fourth Chinese National Arctic Research Expedition of the ‘‘XUELONG’’ icebreaker in July 2010. The sampling site BN12 was located at Makarov basin (W170°290 , N87°040 ) in the Arctic Ocean, with the depth of 4,000 m. The sediment core was firstly sampled by a box sampler, then subsampled by a push core sampler prior to releasing the box corer on deck. Subsequently, the core was sliced into layers at depth intervals of 2 cm, and then about 5 g sediment of each layer was used to set up PAH-degrading consortia. The strain D104T was isolated from the consortium of subsurface sediment layer (8–12 cm) on 216L medium as described elsewhere (Lai et al. 2009). The plates were incubated at 25 °C 2 weeks. The routine cultivation of the strain D104T and most
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The 16S rRNA gene sequence of strain D104T was determined from the draft genome (Dong et al. 2014). The 16S rRNA gene was also amplified by PCR using primers described previously (Liu and Shao 2005). Sequence similarity of 16S rRNA gene was determined using the EzTaxon-e server (Kim et al. 2012). Sequences of 23 strains of related taxa were obtained from the GenBank database. Phylogenetic analysis was performed using MEGA version 5.0 (Tamura et al. 2011). Distances (distance options according to the Kimura two-parameter model) and clustering according to the neighbour-joining (NJ) method of Saitou and Nei (1987), maximum likelihood (ML) (Felsenstein 1981) and minimum evolution (ME) method of Rzhetsky and Nei (1993) were determined by using bootstrap values based on 1,000 replications.
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Genome sequencing, G ? C content, average nucleotide identity (ANI) and DNA–DNA hybridization (DDH) estimation The genome of M. ushuaiensis DSM 15871T was sequenced at Shanghai Majorbio Bio-pharm Technology Co., Ltd (Shanghai, China) using Solexa pairedend (500 bp library) sequencing technology. A total of 500 Mbp clean data for M. ushuaiensis DSM 15871T were generated to reach about 100-fold depth of coverage using an Illumina/Solexa Genome Analyzer IIx (Illumina, SanDiego, CA). The genome was assembled using SOAPdenovo2 (Luo et al. 2012). The accession number of M. ushuaiensis DSM 15871T is JAMB00000000. The draft genome sequence of the strain D104T (AYOZ00000000) was obtained from NCBI (Dong et al. 2014). The G ? C content of the chromosomal DNA was calculated on the basis of its whole genome sequence. The ANI was calculated using J-specises (V1.2.1) as described elsewhere (Richter and Rossello-Mora 2009). DNA–DNA hybridization (DDH) value was estimated using the genome-to-genome distance calculator (GGDC2.0) (Auch et al. 2010a, b; Meier-Kolthoff et al. 2013).
451 Table 1 Physiological characteristics of Marinomonas profundimaris D104T and related species of the genus Marinomonas Characteristic
1
2
3
Oxidase
–
–
?
Growth at: 4 °C
Strain D104T was found to be Gram-negative, aerobic, motile by means of a single flagellum and rod-shaped with the size of 0.6–0.7 lm width and 1.5–1.6 lm length (Supplementary Fig S1). Colonies on marine agar
–
–
?
40 °C
–
–
?
44.8a
43.6 (41.1a)
46.3
API 20NE Arginine dihydrolase
w
?
-
ß-galactosidase
w
-
?
ß-glucosidase (Aesculin hydrolysis)
?
-
w
Trisodium citrate
?
-
?
Urease
–
?
-
Utilization of L-arabinose, N-acetyl-glucosamine
?
-
-
Utilization of malic acid, potassium gluconate Utilization of D-maltose
?
-
?
?
w
?
Esterase(C4), achymotrypsin, naphtol-ASBl-phosphoamidase
–
?
?
Esterase lipase (C8), trypsin
–
?
w
a-galactosidase
–
w
–
b-galactosidase
?
–
–
a-glucosidase, b-glucosidase
?
–
?
w
w
?
Acetoin production (Voges Proskauer)
-
?
-
Tryptophane deaminase
?
w
?
Oxidation arabinose
?
-
?
Oxidation inositol
?
-
w
Oxidation mannitol
?
-
-
Oxidation melibiose, saccharose, sorbitol Susceptibility to:
?
-
w
S
R
S
Enzymatic activity (API ZYM)
Cystine aminopeptidase API 20E
Ampicillin (10 lg), erythromycin (15 lg), penicillin G (10 lg)
Results and discussion Phenotypic characteristics
?
?
DNA G ? C content (mol%)
Determination of fatty acid, isoprenoid ubiquinone and polar lipid compositions The whole cells grown on marine agar 2,216 plate at 25 °C for 48 h were harvested. Fatty acids were saponified, methylated and extracted using the standard protocol of MIDI (Sherlock Microbial Identification System, version 6.0B). The fatty acids were identified by using the TSBA6.0 database of the Microbial Identification System (Sasser 1990). M. ushuaiensis DSM 15871T and M. communis DSM 5604T were tested at the same time. Analysis of respiratory quinone and polar lipid were carried out by the Identification Service of the DSMZ, Braunschweig, Germany.
?
35 °C
Strains: 1, D104T; 2, M. ushuaiensis DSM 15871T (Prabagaran et al. 2005); 3, M. communis DSM 5604T (Chimetto et al. 2011) API 20NE, API 20E, API ZYM and susceptibility to antibiotics data are performed in this study. Characteristics are scored as: ? positive, - negative, w weak, S sensitive, R resistive a
Data from draft genome sequence
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Marinomonas arctica 328T (DQ492749) Marinomonas hwangdonensis HDW-15T (JN087530) Marinomonas polaris CK13T (AJ833000) Marinomonas primoryensis KMM 3633T (AB074193) Marinomonas rhizomae IVIA-Po-145T (EU188443)
Marinomonas foliarum IVIA-Po-155T (EU188444) Marinomonas alcarazii IVIA-Po-14bT (EU188442)
77 98
Marinomonas pontica 46-16T (AY539835)
Marinomonas profundimaris D104T (KC565667) Marinomonas ushuaiensis U1T (AJ627909) 95 Marinomonas arenicola KMM 3893T (AB467281) 97 Marinomonas pollencensis IVIA-Po-185T (EU188441) Marinomonas dokdonensis DSW10-10T (DQ011526) Marinomonas posidonica IVIA-Po-181T (CP002771) 97 100 Marinomonas aquiplantarumi IVIA-Po-159T (EU188447) Marinomonas brasilensis R-40503T (GU929940) Marinomonas vaga ATCC 27119T (X67025)
63
Marinomonas communis LMG 2864T (DQ011528) Marinomonas aquimarina CECT 5080T (AJ843077)
65 95
100
Marinomonas fungiae AN44T (JQ409370) Marinomonas ostreistagni UST010306-043T (AB242868) Marinomonas mediterranea MMB-1T (CP002583) Marinomonas balearica IVIA-Po-101T (EU188448) Marinobacterium lutimaris AN9T (FJ230839)
0.01 Fig. 1 Neighbour-joining tree showing the phylogenetic position of Marinomonas profundimaris D104T within genus Marinomonas based on 16S rRNA gene sequence similarities. Filled circles indicate nodes that were also recovered in maximum-likelihood and minimum evolution trees based on
the same sequences. Bootstrap values (expressed as percentages of 1,000 replications) are shown at branch points. Bar, 0.01 nucleotide substitution rate (Knuc) units. Marinobacterium lutimaris AN9T was used as the outgroup
2,216 are round, dull white, non-pigmented, nonluminescent with irregular margins and 2–3 mm in diameter after 48 h incubation at 25 °C. Growth occurs at 4–37 °C (optimum 25–28 °C) and pH 5–10 (optimum 7) and in presence 0.5–6 % of NaCl (optimum 3 %). Strain D104T is found to be sensitive to ampicillin (10 lg/per disk, Oxoid), carbenicillin (100), cefalexin (30), cefazolin (30), chloromycetin (30), ciprofloxacin (5), erythromycin (15), furazolidone (15), furazolidone (5), gentamicin (10), kanamycin (30), minomycin (30), neomycin (10), norfloxacin (10), ofloxacin (5), penicillin G (10), polymyxin B (30 IU), streptomycin (10),
tetracycline (30), vibramycin (30); resistant to clindamycin (2), lincomycin (2), metronidazole (5), oxacillin (1), piperacillin (100) and vanoomycin (30). Other characteristics are given in species description. The characteristic phenotypic features of strain D104T and two reference type strains are shown in Table 1.
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Phylogenetic analysis based on 16S rRNA gene sequence similarities A nearly full-length 16S rRNA gene sequence (1,503 nt of strain D104T was used in phylogenetic
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analysis. As shown in Fig. 1, phylogenetic tree based on 16S rRNA gene sequence similarities showed that strain D104T formed a robust clade with M. ushuaiensis DSM 15871T within the genus Marinomonas (95 % bootstrap support). Same topology was retrieved in maximum-likelihood and minimum evolution trees (Supplementary materials Figs. S2 and S3). Strain D104T was found to have the highest sequence similarity of 97.7 % to M. ushuaiensis DSM 15871T, followed by M. dokdonensis DSW10-10T (96.9 %), M. arenicola KMM 3893T (96.7 %), M. arctica 328T (96.6 %) and other 18 species of the genus Marinomonas (94.4–96.5 %). DNA G ? C content, ANI and DDH The DNA G ? C content of strain D104T was found to be 44.8 mol% which is in the range of the genus Marinomonas (i.e., 41–50 mol%) (Espinosa et al. 2010). The DNA G ? C content of M. ushuaiensis DSM 15871T was estimated to be 41.3 mol% according to its draft genome sequence, which was comparable to previously reported value of 43.6 mol%. The ANI of strain D104T and M. ushuaiensis DSM 15871T was found to be 84.24 %. This value is significantly lower than the suggested threshold range of 95–96 % (Richter and Rossello-Mora 2009). The DDH estimated value between two strains was found to be 20.80 ± 2.33 %, which is also significantly lower than the recommended threshold of 70 % (Wayne et al. 1987). These results suggested that strain D104T represents a novel genomic species of the genus Marinomonas. Chemotaxonomic characteristics The principal fatty acids of strain D104T were found to be sum in feature 3 (C16:1x7c/C16:1x6c) (27.1 %), sum in feature 8 (C18:1x7c/C18:1x6c) (39.7 %), C16:0 (12.3 %) and C12:1 3OH (5.8 %) (Table S1). The fatty acids patterns of all tested strains appeared to be similar with the same characteristics fatty acids ([10 %) and C10:0 3OH ([2 %) (Espinosa et al. 2010). The respiratory quinone of strain D104T was found to be Q-8 (100 %) in agreement with previously reported data (Yoon et al. 2005; Jung et al. 2012). Polar lipids of strain D104T were found to consist of a phosphatidylglycerol (PG), a phosphatidylethanolamine (PE), an aminolipid (AL) and a phospholipid (PL) (Supplementary Fig. S4). The major polar lipid,
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PG and PE, were also present in type species M. communis and other species of the genus Marinomonas (Gupta et al. 2006; Zhang et al. 2008; Jung et al. 2012). Taxonomic conclusion Strain D104T could be assigned to the genus Marinomonas based on the results of phylogenetic analysis, phenotypic analysis and chemotaxonomic data. However, the strain D104T could be distinguished from the close phylogenetic relatives by several phenotypic characteristics listed in Table 1. On the basis of the results presented in this study and taking into account DNA–DNA hybridization values below 70 % it is proposed that strain D104T can be classified as novel species of the genus Marinomonas for which the name Marinomonas profundimaris sp. nov. is proposed. Description of Marinomonas profundimaris sp. nov Marinomonas profundimaris (pro.fun.di.ma’ris. L. adj. profundus deep; L. gen. n. maris from/of the sea; N.L. gen. n. profundimaris from/of the deep-sea, reflecting from where the type strain was isolated). Cells are Gram-negative, rod-shaped, about 0.6–0.7 lm width and 1.5–1.6 lm length, motile with a single polar flagellum. Aerobic. Chemoorganotroph with respiratory metabolism. The colonies are round, dull white and non-pigmented with irregular margins, 2–3 nm in diameter, convex, smooth on marine agar. Catalase weakly positive, oxidase negative. Negative for Tween 80 and casein hydrolysis, positive for amylase. Requires NaCl for growth. Growth occurs in media with 0.5–6 % of NaCl (optimum 3 %). Temperature for growth ranges from 4 to 37 °C (optimum 25-28 °C). The pH for growth ranges from 5 to 10 (optimum pH 7). According to API ZYM, positive for acid phosphatase, alkaline phosphatase, leucine aminopeptidase, valine aminopeptidase, a-glucosidase, bgalactosidase, b-glucosidase and weakly positive for cystine aminopeptidase, lipase (C14); negative for bglucuronidase, esterase lipase (C8), esterase (C4), N-acetyl-b-glucosaminidase, naphtol-AS-Bl-phosphoamidase, trypsin, a-chymotrypsin, a-fucosidase, a-galactosidase, a-mannosidase. According to API 20NE, positive for arginine dihydrolase, b-galactosidase (weak), b-glucosidase (aesculin hydrolysis) and utilization of D-glucose, D-mannitol, D-mannose,
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L-arabinose, malic acid, potassium gluconate and trisodium citrate; negative for D-glucose fermentation, gelatinase, urease, indole production, nitrate reduction and utilization of capric acid, phenylacetic acid, adipic acid, D-maltose and N-acetyl-glucosamine. According to API 20E, positive for citrate, tryptophane deaminase, arginine dihydrolase (weak), ß-galactosidase (weak) and oxidation of arabinose, inositol, mannitol, melibiose, saccharose, sorbitol; negative for gelatinase, H2S production, indole production, lysine decarboxylase, ornithine decarboxylase and oxidation of amygdalin, glucose and rhamnose. According to Biolog GN2 system, utilizes bromo succinic acid, cis-aconitic acid, citric acid, D,L-lactic acid, D-arabitol, D-cellobiose, D-fructose, D-galactose, D-gluconic acid, D-mannitol, D-mannose, D-melibiose, D-sorbitol, Dtrehalose, glucose-6-phosphate, glycerol, glycyl-Lglutamic acid, inosine, L-asparagine, L-aspartic acid, L-glutamic acid, L-ornithine, L-proline, maltose, m-inositol, succinic acid, sucrose, turanose, uridine, urocanic acid, a-D-glucose, a-keto glutaric acid and c-amino butyric acid; weakly positive for 2,3-butanediol, 2-aminoethanol, acetic acid, D,L-a-glycerol phosphate, D-alanine, dextrin, D-galacturonic acid, D-glucuronic acid, D-raffinose, D-saccharic acid, glucose-1-phosphate, glycogen, glycyl-L-aspartic acid, L-alanine, L-alanyl-glycine, L-pyroglutamic acid, L-serine, malonic acid, methyl pyruvate, mono-methylsuccinate, N-acetyl-D-glucosamine, putrescine, quinic acid, succinamic acid, thymidine, tween 40, tween 80, b-hydroxy butyric acid, c-hydroxy butyric acid; negative for others. The main cellular fatty acids are sum in feature 3 (C16:1x7c/C16:1x6c), sum in feature 8 (C18:1x7c/C18:1x6c), C16:0 and C12:1 3OH. The respiratory quinone is Q-8 (100 %). Phosphatidylglycerol (PG), phosphatidylethanolamine (PE) and aminolipid (AL) are main polar lipids. The G ? C content of the chromosomal DNA of the type strain is 44.8 mol%. The type strain D104T (= MCCC 1A07573T = LMG 27696T), isolated from deep sea sediment sample from Arctic Ocean. The GenBank accession number of the genome is AYOZ00000000. Acknowledgments This work was financially supported by Public Welfare Project of SOA (201005032), COMRA program (No. DY125-15-R-01), National Natural Science Foundation of China (41206158), the China Polar Environment Investigation and Estimate Project (2012-2015) and National Infrastructure of Natural Resources for Science and Technology Program of China (No. NIMR-2013-9).
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