Marinobacter flavimaris sp. nov. and Marinobacter daepoensis sp. nov [PDF]

type strains of all Marinobacter species. Levels of DNA–DNA relatedness, together with 16S rRNA gene sequence similari

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International Journal of Systematic and Evolutionary Microbiology (2004), 54, 1799–1803

DOI 10.1099/ijs.0.63151-0

Marinobacter flavimaris sp. nov. and Marinobacter daepoensis sp. nov., slightly halophilic organisms isolated from sea water of the Yellow Sea in Korea Jung-Hoon Yoon,1 Soo-Hwan Yeo,2 In-Gi Kim1 and Tae-Kwang Oh1 Correspondence Jung-Hoon Yoon [email protected]

1

Korea Research Institute of Bioscience and Biotechnology (KRIBB), PO Box 115, Yusong, Taejon, Korea

2

The Centre for Traditional Micro-organism Resources, Keimyung University, Shindang-Dong, Dalseo-gu, Daegu, Korea

Two Gram-negative, motile, non-spore-forming and slightly halophilic rods (strains SW-145T and SW-156T) were isolated from sea water of the Yellow Sea in Korea. Strains SW-145T and SW-156T grew optimally at 37 and 30–37 6C, respectively, and in the presence of 2–6 % (w/v) NaCl. Strains SW-145T and SW-156T were chemotaxonomically characterized as having ubiquinone-9 as the predominant respiratory lipoquinone and C16 : 0, C18 : 1v9c, C16 : 1v9c and C12 : 0 3-OH as the major fatty acids. The DNA G+C contents of strains SW-145T and SW-156T were 58 and 57 mol%, respectively. Phylogenetic analyses based on 16S rRNA gene sequences showed that strains SW-145T and SW-156T fell within the evolutionary radiation enclosed by the genus Marinobacter. The 16S rRNA gene sequences of strains SW-145T and SW-156T were 94?8 % similar. Strains SW-145T and SW-156T exhibited 16S rRNA gene sequence similarity levels of 94?3–98?1 and 95?4–97?7 %, respectively, with respect to the type strains of all Marinobacter species. Levels of DNA–DNA relatedness, together with 16S rRNA gene sequence similarity values, indicated that strains SW-145T and SW-156T are members of two species that are distinct from seven Marinobacter species with validly published names. On the basis of phenotypic properties and phylogenetic and genotypic distinctiveness, strains SW-145T (=KCTC 12185T=DSM 16070T) and SW-156T (=KCTC 12184T=DSM 16072T) should be placed in the genus Marinobacter as the type strains of two distinct novel species, for which the names Marinobacter flavimaris sp. nov. and Marinobacter daepoensis sp. nov. are proposed.

The genus Marinobacter was proposed by Gauthier et al. (1992) with a single species, Marinobacter hydrocarbonoclasticus. The second species, Marinobacter aquaeolei, was described by Huu et al. (1999). Recently, five further species, Marinobacter litoralis (Yoon et al., 2003), Marinobacter lipolyticus (Martı´n et al., 2003), Marinobacter excellens (Gorshkova et al., 2003), Marinobacter lutaoensis (Shieh et al., 2003) and Marinobacter squalenivorans (Rontani et al., 2003), have been added to the genus Marinobacter. Members of the genus Marinobacter have been isolated from marine environments, saline soil and coastal hot springs. In this study, we describe two Gram-negative, slightly halophilic strains, SW-145T and SW-156T, isolated from sea water at Daepo Beach of the Yellow Sea in Korea. The two organisms The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains SW-145T and SW-156T are AY517632 and AY517633, respectively. Details of the fatty acid compositions of the novel strains and related species are available as supplementary material in IJSEM Online.

63151 G 2004 IUMS

were considered to be Marinobacter-like strains on the basis of 16S rRNA gene sequence comparisons. Accordingly, the aim of the present study was to determine the exact taxonomic positions of strains SW-145T and SW-156T by means of a polyphasic characterization that included determination of phenotypic properties and a detailed phylogenetic analysis based on 16S rRNA gene sequences and genotypic relatedness. Strains SW-145T and SW-156T were isolated by using the dilution plating technique on marine agar 2216 (MA) (Difco). Cell morphology was examined using light microscopy (Nikon E600) and transmission electron microscopy. The latter was used to examine flagellum type in cells from exponentially growing cultures. The Gram reaction was determined using the bioMe´rieux Gram stain kit according to the manufacturer’s instructions. Growth under anaerobic conditions was determined after incubation in an anaerobic chamber using MA and MA supplemented with nitrate, both of which had been prepared anaerobically.

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Printed in Great Britain

1799

J.-H. Yoon and others

Growth at various NaCl concentrations was investigated in marine broth 2216 (MB) (Difco) or trypticase soy broth (Difco). Growth in the absence of NaCl was investigated in trypticase soy broth lacking NaCl. Growth at various temperatures (4–50 uC) was measured on MA. Catalase and oxidase activities and hydrolysis of casein, starch and Tweens 20, 40, 60 and 80 were determined as described by Cowan & Steel (1965). Hydrolysis of aesculin, gelatin and urea and nitrate reduction were determined as described previously (Lanyi, 1987) with the modification that artificial sea water was used. The artificial sea water contained (l21 distilled water) 23?6 g NaCl, 0?64 g KCl, 4?53 g MgCl2.6H2O, 5?94 g MgSO4.7H2O and 1?3 g CaCl2.2H2O (Levring, 1946). Hydrolysis of hypoxanthine, tyrosine and xanthine was tested on MA plates using the substrate concentrations described previously (Cowan & Steel, 1965). H2S production was tested as described previously (Bruns et al., 2001). The API ZYM system (bioMe´rieux) was used to determine the activity of some enzymes. Acid production from carbohydrates was determined as described by Leifson (1963). Tests for the utilization of various substrates were performed as described previously (Yurkov et al., 1994). Cell biomass for respiratory lipoquinone analysis and for DNA extraction was obtained from MB cultures at 30 uC. M. hydrocarbonoclasticus DSM 8798T, M. aquaeolei DSM 11845T, M. litoralis KCCM 41591T, M. lipolyticus SM19T and M. excellens KMM 3809T were used as reference strains for DNA–DNA hybridization. Cell mass for DNA extraction from reference strains was obtained from MB cultures at 30 uC. Respiratory lipoquinones were analysed as described previously (Komagata & Suzuki, 1987) using reversedphase HPLC. Chromosomal DNA was isolated and purified according to the method described previously (Yoon et al., 1996), with the exception that ribonuclease T1 was used together with ribonuclease A. The DNA G+C content was determined by the method of Tamaoka & Komagata (1984). DNA was hydrolysed and the resultant nucleotides were analysed by reversed-phase HPLC. For fatty acid methyl ester analysis, a loop of cell mass of strain SW-145T and of strain SW-156T was harvested from agar plates after incubation for 3 days at 30 uC on MA. The fatty acid methyl esters were extracted and prepared according to the standard protocol of the MIDI/Hewlett Packard Microbial Identification System (Sasser, 1990). 16S rRNA genes were amplified by a PCR using two universal primers as described previously (Yoon et al., 1998). Sequencing of the amplified 16S rRNA genes and phylogenetic analysis were performed as described by Yoon et al. (2003). DNA– DNA hybridization was performed fluorometrically by the method of Ezaki et al. (1989) using photobiotin-labelled DNA probes and microdilution wells. Hybridization was performed with five replications for each sample. The highest and lowest values obtained in each sample were excluded and the remaining three values were used to calculate relatedness values. The DNA relatedness values quoted are the means of the three values. 1800

Strains SW-145T and SW-156T were similar in terms of most morphological, cultural, physiological and biochemical characteristics. However, strain SW-145T grew at 4 uC, whereas strain SW-156T did not. Strain SW-145T grew in the presence of 20 % NaCl, but strain SW-156T did not. D-Fructose, glycerol and D-gluconate were utilized by strain SW-145T, but not by strain SW-156T. There were also differences in lipase activity (C14) and nitrate reduction between the two strains (Table 1). Strains SW-145T and SW-156T are differentiated from other Marinobacter species by means of some phenotypic characteristics, including tolerance of NaCl, temperature and pH for growth and the ability to utilize some substrates (Table 1). Other phenotypic characteristics are shown in Table 1 or are given in the species descriptions. Strains SW-145T and SW-156T contained ubiquinone-9 (approx. 85 and 96 %, respectively) as the predominant respiratory lipoquinone. Strains SW-145T and SW-156T had cellular fatty acid profiles containing large amounts of straight-chain, unsaturated and hydroxyl fatty acids. The major fatty acids detected in strain SW145T were C16 : 0 (26?7 %), C18 : 1v9c (17?4 %), C16 : 1v9c (10?2 %), C12 : 0 3-OH (10?5 %) and C12 : 0 (9?1 %). Strain SW-156T contained the following major fatty acids: C16 : 0 (24?8 %), C18 : 1v9c (24?3 %), C16 : 1v9c (12?8 %), C12 : 0 3-OH (9?4 %) and C12 : 0 (7?1 %). These profiles were similar to those of the type strains of Marinobacter species described previously, although there were some differences in the compositions of some fatty acids (see the supplementary table in IJSEM Online). Two fatty acids, C16 : 1v7c and C19 : 0, were detected in M. excellens KMM 3809T, but not in strains SW-145T, SW-156T or other Marinobacter species. However, the two fatty acids were detected from the type strains of M. hydrocarbonoclasticus, M. aquaeolei and M. litoralis in the study by Gorshkova et al. (2003). This observation may have been caused by different experimental conditions, e.g. cultivation conditions or analytical equipment. The predominant respiratory lipoquinone and the cellular fatty acid profiles of strains SW-145T and SW156T are consistent with those of Marinobacter species except M. lutaoensis (Yoon et al., 2003; Gorshkova et al., 2003; Shieh et al., 2003). M. lutaoensis contained ubiquinone-8 as the predominant ubiquinone and iso-C15 : 0 as the major fatty acid (Shieh et al., 2003). The DNA G+C contents of strains SW-145T and SW-156T were 58 and 57 mol%, respectively. Almost complete 16S rRNA gene sequences of strains SW-145T and SW-156T comprising, respectively, 1495 and 1496 nt (approx. 96 % of the Escherichia coli 16S rRNA gene sequence) were determined in this study. Strains SW-145T and SW-156T were found to have the highest 16S rRNA gene sequence similarity values to members of the c-Proteobacteria. The level of 16S rRNA gene sequence similarity between strains SW-145T and SW-156T was 94?8 %. A neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showed that strains SW-145T and SW-156T fell within the radiation of the cluster comprising Marinobacter species (Fig. 1). Similar tree topologies were

Downloaded from www.microbiologyresearch.org by International Journal of Systematic and Evolutionary Microbiology 54 IP: 23.229.114.237 On: Thu, 04 Apr 2019 20:53:45

Two novel Marinobacter species

Table 1. Differential phenotypic characteristics of strains SW-145T and SW-156T and other Marinobacter species Strains/species: 1, strain SW-45T; 2, strain SW-56T; 3, M. hydrocarbonoclasticus (data from Gauthier et al., 1992; Yoon et al., 2003); 4, M. aquaeolei (Huu et al., 1999); 5, M. litoralis (Yoon et al., 2003); 6, M. lipolyticus (Martı´n et al., 2003); 7, M. excellens (Gorshkova et al., 2003); 8, M. lutaoensis (Shieh et al., 2003). +, Positive; 2, negative; W, weak; ND, not determined. The following tests were positive for all species: motility, catalase, oxidase, alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, acid phosphatase, naphtholAS-BI-phosphohydrolase and N-acetyl-b-glucosaminidase. The following tests were negative for all species: cystine arylamidase, trypsin, achymotrypsin, a-galactosidase, b-galactosidase, b-glucuronidase, a-glucosidase, b-glucosidase, a-mannosidase, a-fucosidase and utilization of D-cellobiose, D-mannose and sucrose. Characteristic Flagellation Urease Lipase (C14)* Valine arylamidase* Nitrate reduction to nitrite Nitrite reduction to N2 Hydrolysis of: Aesculin Gelatin Starch Tween 80 Growth at/in: pH 5?0 0 % NaCl 20 % NaCl 4 uC Utilization of: D-Glucose D-Fructose D-Mannitol L-Glutamate D-Gluconate Succinate DL-Alanine L-Arginine Aspartate Temperature for growth (uC): Maximum Optimum Predominant ubiquinone DNA G+C content (mol%)

1

2

3

4

5

6

7

8

Single polar 2 + 2 + 2

Single polar 2 2 2 2 2

Single polar or none 2 2

Single polar + 2 + + 2

Single polar + 2 2 + 2

ND

Single polar

Single or several polar

ND

ND

ND

ND

ND

ND

ND

ND

ND

2 2

+ +

2 2

2 2 2 +

2 2 2 +

ND

2 +

ND

ND

ND

2 2 +

2 + +

2 2 ND

2 2 + +

+ 2 2 2

W

+ +

2 2

ND

ND

+

2 2 2 +

2 2 2 2

2 2 + 2

+ + + 2

+ 2 2 +

+ 2 2 2

2 2 2 2

2 + 2 2 + + 2 2 2

2 2 2 2 2 + 2 2 2

2 2 2 + 2 + 2 2 2

2 2 2 + 2 + 2 2 2

2 2 2 + 2 + 2 2 2

+ + + 2 + 2 2

2 + + 2 2 + 2

ND

ND

2

2

+ (L-) 2 +

45 37 Q-9 58

45 30–37 Q-9 57

45 32 Q-9 53

50 30 Q-9 56

46 30–37 Q-9 55

40 37

41 20–25 Q-9 55–56

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