Antonie van Leeuwenhoek

, Volume 101, Issue 1, pp 133–140 | Cite as

Tunicatimonas pelagia gen. nov., sp. nov., a novel representative of the family Flammeovirgaceae isolated from a sea anemone by the differential growth screening method

  • Jaewoo Yoon
  • Naoya Oku
  • Sanghwa Park
  • Atsuko Katsuta
  • Hiroaki Kasai
Original Paper

Abstract

A Gram-negative, strictly aerobic, reddish-pink pigmented, non-motile, rod-shaped strain designated N5DB8-4T, was isolated from an orange-striped sea anemone Diadumene lineata by a differential growth screening method. Phylogenetic analyses based on the 16S rRNA gene sequence revealed that the novel isolate was affiliated with the family Flammeovirgaceae of the phylum Bacteroidetes and that it showed highest sequence similarity (89.1%) to Porifericola rhodea N5EA6-3A2BT. The strain could be differentiated phenotypically from recognized members of the family Flammeovirgaceae. The G+C content of the DNA is 52.6 mol%, the major respiratory quinone is menaquinone 7 (MK-7) and iso-C15:0, C16:1ω5c and iso-C15:1 G (the double-bond position indicated by capital letter is unknown) were the major fatty acids. From the distinct phylogenetic position and combination of genotypic and phenotypic characteristics, the strain represents a novel taxon for which the name Tunicatimonas pelagia gen. nov., sp. nov. is proposed. The type strain of Tunicatimonas pelagia is N5DB8-4T (=KCTC 23473= NBRC 107804T).

Keywords

Tunicatimonas pelagia gen. nov., sp. nov. Concanavalin A Differential growth screening method 16S rRNA gene Polyphasic taxonomy 

Introduction

The phylum Bacteroidetes (previously known as the Cytophaga-Flavobacterium-Bacteroides or CFB group) is one of the major phylogenetic lineages in the domain Bacteria. They are widely distributed in the global ecosystems such as soil, sediments and seawater (O’Sullivan et al. 2002; Kirchman 2002). Remarkably, members of the phylum Bacteroidetes constitute a significant proportion of marine microbial communities (Glöckner et al. 1999) and are associated with the decomposition of organic biomacromolecules (Cottrell and Kirchman 2000) and planktonic blooms (Brettar et al. 2004). Also, several species in this phylum are pathogenic to humans and animals (Bernardet et al.2002). Currently, a classification of members within the phylum Bacteroidetes is still in a state of development. In the second edition of Bergey’s Manual of Systematic Bacteriology (volume 4), the family Flammeovirgaceae was proposed within this phylum (Family III. Flammeovirgaceae fam. nov.). It includes the genera Aureibacter, Flammeovirga, Fabibacter, Flexithrix, Fulvivirga, Limibacter, Marinoscillum, Perexilibacter, Persicobacter, Rapidithrix, Reichenbachiella, Roseivirga, Sediminitox and several generically misclassified species. The formal description of the family Flammeovirgaceae (Yoon et al. 2010) comprised the genera Aureibacter (Yoon et al. 2010), Flammeovirga (Nakagawa et al. 1997), Flexithrix (Lewin 1970), Limibacter (Yoon et al. 2008), Perexilibacter (Yoon et al. 2007), Persicobacter (Nakagawa et al. 1997), and Sediminitox (Khan et al. 2007). Here, we performed a polyphasic taxonomic characterization of a novel marine member (strain N5DB8-4T) of the family using a polyphasic taxonomic approach following its isolation from the sea anemone Diadumene lineata by employing a differential growth screening method.

Materials and methods

Isolation of bacterial strains and culture conditions

Strain N5DB8-4T was one of 13 isolates whose growth was dependent on concanavalin A, a d-glucose- and d-mannose-specific lectin from the jack bean Canavalia ensiformis. The isolates were identified by differential growth screening of 364 isolates obtained with concanavalin A-supplemented medium from 47 marine-derived samples collected along the eastern coast of Tokyo Bay, Japan. The samples included 29 marine animals, seven sea sediments, five seaweeds, four animal remains and two pieces of driftwood.

The collected specimens were cut into pieces with sterile scissors and sections approximately 1 cm3 in size were placed into a 15-ml polypropylene centrifuge tube containing 5 ml of sterile seawater. The contents were crushed vigorously with a glass rod and the resulting tissue suspensions were left to stand for an hour. The supernatants were then diluted 1/100 with sterile seawater and 50 μl aliquots were spread over the isolation media, designated as 1/10 MA-Con.A [marine broth 2216 (Difco) 5.51 g/l, agar 15 g/l, and 75% natural seawater; supplemented with powdered concanavalin A 10 mg/l after the autoclaved mixture was cooled to 80°C]. The plates were left at ambient temperature (20–28°C) for a week and colonies were transferred to the same medium for further studies. Inocula made from these isolates were smeared onto unsupplemented 1/10 MA to examine their growth without the plant lectin. Fifty-four of the 364 isolates did not grow within two weeks. Finally, a simultaneous comparison of the aforementioned two media was made with these strains to confirm their dependency on the lectin. Sixteen grew well on both media and were classified as concancavalin A independent, while 13 showed remarkable differences in growth. The remaining 25 isolates did not grow, even in the presence of the lectin.

Strain N5DB8-4T was originally isolated from an orange-striped sea anemone Diadumene lineata (formerly Haliplanella lineate, family Diadumenidae, order Actiniaria) that inhabited the remains of an oyster shell that was encrusted on a concrete block revetment along a reclaimed waterfront park in Sodegaura, Chiba (35°27′17.0″ N, 139°57′63.8″ E). Strain N5DB8-4T formed tiny red colonies on 1/10 MA-Con.A but failed to grow on 1/10 MA. However, during subsequent experiments, it was found to grow without any problems on normal strength marine agar 2216 (Difco).

The strain was routinely cultured on marine agar 2216 at 25°C and maintained in marine broth 2216 supplemented with 20% (v/v) glycerol at –70°C.

Morphological, physiological and biochemical analysis

Cell morphology was observed using light microscopy (BX60; Olympus) and flagella staining was carried out according to Blenden and Goldberg (1965). Gliding motility was determined as described by Perry (1973). The temperature (4–45°C) and pH (5–10) ranges for growth were determined by incubating the isolates on marine agar 2216. The NaCl concentration for growth was determined on marine agar 2216 containing 0–10% (w/v) NaCl (Atlas 1993). Gram-staining was performed as described by Murray et al. (1994). Anaerobic growth was tested for up to 2 weeks on marine agar 2216 in a jar containing AnaeroPack-Anaero (Mitsubishi Gas Chemical Co., Inc.), which works as an O2 absorber and CO2 generator. Catalase activity was determined by bubble formation in a 3% H2O2 solution. Oxidase activity was determined using cytochrome oxidase paper (Nissui Pharmaceutical Co., Inc.). Degradation of DNA was tested using DNase agar (Scharlau), with DNase activity detected by flooding plates with 1 M HCl. Starch hydrolysis were tested as described by Choi et al. (2007). The ability to hydrolyse casein, Tweens 20 and 80 and tyrosine were determined according to Hansen and Sørheim (1991). API 20E, API 50CH and API ZYM strips (bioMérieux) were used to determine the physiological and biochemical characteristics. All suspension media for the API test strips were supplemented with 0.85% (w/v) NaCl solution (final concentration). API 20E and API 50CH test strips were read after 72 h incubation at 30°C and API ZYM test strips were read after 4 h incubation at 37°C. Flexirubin-type pigments were investigated by using the bathochromatic shift test with a 20% (w/v) KOH solution (Bernardet et al. 2002).

Determination of G+C content of DNA, 16S rRNA gene sequencing and phylogenetic analysis

Genomic DNA was prepared according to the method of Marmur (1961) from cells grown on marine agar 2216 and the DNA base composition was determined by using the HPLC method of Mesbah et al. (1989). An approximately 1,500 bp fragment of the 16S rRNA gene was amplified from the extracted DNA by using bacterial universal primers specific to the 16S rRNA gene: 27F and 1,492R (Escherichia coli numbering system; Weisburg et al. 1991). To ascertain the phylogenetic position of the novel isolate, the 16S rRNA gene sequence of strain N5DB8-4T (GenBank/EMBL/DDBJ accession number AB602437) was compared with sequences obtained from GenBank (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov). Multiple alignments of the sequences were performed using CLUSTAL_X (version 1.83) (Thompson et al. 1997). Alignment gaps and ambiguous bases were not taken into consideration when 1,242 bases of the 16S rRNA gene were compared. Aligned sequences were analyzed by the MEGA4 software (Tamura et al. 2007). The evolutionary distances [distance options according to the Kimura two-parameter model (Kimura 1983)] and clustering with the neighbor-joining (Saitou and Nei 1987) and maximum-parsimony (Fitch 1971) methods were determined by using bootstrap values based on 1,000 replications (Felsenstein 1985). The similarity values were calculated using the same software.

Chemotaxonomic analysis

Gas chromatography analysis of the cellular fatty acid methyl esters was performed using a culture grown on marine agar 2216 at 27°C for 4 days. Fatty acid methyl esters were extracted and prepared according to standard protocols provided by the MIDI/Hewlett Packard Microbial Identification system (Sasser 1990). Determination of the respiratory quinone system was carried out as described previously (Xie and Yokota 2003).

Results and discussion

Molecular phylogenetic analysis

Molecular phylogenetic analysis based on 16S rRNA gene sequences revealed that strain N5DB8-4T belonged to the family Flammeovirgaceae within the phylum Bacteroidetes and showed highest sequence similarity (89.1% identity) to Porifericola rhodea N5EA6-3A2BT (Yoon et al. 2011). All other recognized species of the family Flammeovirgaceae were more distantly related, showing 16S rRNA gene sequence similarity to strain N5DB8-4T of less than 87% (Fig. 1).
Fig. 1

Neighbour-joining tree of 16S rRNA gene sequence similarity, showing the phylogenetic position of strain N5DB8-4T and representatives of the family Flammeovirgaceae. The sequence of Thermonema lapsum ATCC 43542T was used as an outgroup. The sequence determined in this study is shown in bold. Bootstrap values from both the neighbor-joining (above nodes) and maximum-parsimony (below nodes) analyses are shown. Bar 5% sequence divergence

Morphological, physiological and biochemical characteristics

Cells were straight rods with a length of 15–30 μm, devoid of flagella or appendages and produced a reddish-pink pigment. Gliding motility was not observed by light microscopy and flexirubin-type pigments were not produced (Table 1).
Table 1

Differential characteristics of strain N5DB8-4T and other closely related taxa

Characteristic

1

2

3

4

5

6

7

8

9

Gliding motility

+

+

+

+

+

+

Temperature range for growth (°C)

20–37

20–30

4–39

12–44

14–44

25–28

10–40

15–40

41

Highest NaCl tolerance (%, w/v)

8

10

8

16

3

6

10

5.4

12

Hydrolysis of:

         

 Agar

+

+

 DNA

+

+

+

+

+

 Gelatin

+

+

+

+

+

+

DNA G+C content (mol%)

52.6

43

40.2

42.5

59.9

44.5

36.1

41.5

44–45

Characteristic

10

11

12

13

14

15

16

17

18

Gliding motility

+

+

+

+

+

+

+

Temperature range for growth (°C)

20–37

15–45

35

15–30

15–40

17–37

30–37

25–30

4–33

Highest NaCl tolerance (%, w/v)

5

7

6

5

4

6.5

3.5

ND

6

Hydrolysis of

         

 Agar

+

+

W

ND

 DNA

+

ND

+

+

ND

+

 Gelatin

+

+

ND

+

+

+

+

DNA G+C content (mol%)

36.2

42.6–43.8

37

34.2

38

27.9

43

40

35.5

Strains: 1 N5DB8-4T (Tunicatimonas pelagia gen. nov., sp. nov.; present study), 2Porifericola rhodea N5EA6-3A2BT (Yoon et al. 2011), 3Roseivirga ehrenbergii KMM 6017T (Nedashkovskaya et al. 2005a, b, c), (Nedashkovskaya et al. 2005a, b, c), 4Fabibacter halotolerans UST030701-084T (Lau et al., 2006), 5Fulvivirga kasyanovii KMM 6220T (Nedashkovskaya et al. 2007a, b), 6Reichenbachiella agariperforans (Nedashkovskaya et al. 2003), 7Marivirga tractuosa NBRC 15989T (Nedashkovskaya et al. 2010), 8Marinoscillum pacificum MRN461T (Seo et al. 2009), 9Echinicola pacifica KMM 6172T (Nedashkovskaya et al. 2006), 10Aureibacter tunicatorum A5Q-118T (Yoon et al. 2010), 11Persicobacter diffluens NBRC 15940T (Muramatsu et al. 2010), 12Flexithrix dorothea ATCC 23163T (Lewin 1970), 13 Flammeovirga aprica NBRC 15941T (Takahashi et al. 2006), 14Sediminitomix flava Mok-1-85T (Khan et al. 2007), 15 Limibacter armeniacum YM11-185T (Yoon et al. 2008), 16 Perexilibacter aurantiacus Shu-F-UV2-2T (Yoon et al. 2007), 17Rapidithrix thailandica TISTR 1750T (Srisukchayakul et al. 2007), 18Belliella baltica BA134T (Brettar et al. 2004)

+ positive, − negative, W weakly positive, ND no data

Chemotaxonomic characteristics

As shown in Table 2, the predominant cellular fatty acids of strain N5DB8-4T were iso-C15:0 (24.5%), C16:1ω5c (21.3%) and iso-C15:1 G (The double-bond position indicated by capital letter is unknown; 11.3%). On the basis of the fatty acid composition, strain N5DB8-4T could be differentiated from the phylogenetically closest taxa such as P. rhodea N5EA6-3A2BT, Fulvivirga kasyanovii KMM 6220T and Marivirga tractuosa NBRC 15989T as shown in Table 2. Additionally, the strain could also be distinguished from the neighboring taxa by the presence of iso-C13:0 (9.8%), and iso-C15:1 G (11.3%) and the absence of anteiso-C15:0, iso-C16:0, and iso-C16:0 3-OH. From these results, it is strongly suggested that strain N5DB8-4T represents an independent genus of the family Flammeovirgaceae within the phylum Bacteroidetes.
Table 2

Comparison of cellular fatty acids for strain N5EA6-3A2BT and other closely related taxa

Fatty acid

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

iso-C13:0

9.8

3.2

1.6

1.2

tr

iso-C14:0

tr

1

4.7

tr

tr

0.1–0.2

1.8

2

iso-C15:1 G

11.3

4.9

tr

iso-C15:0

24.5

34.4

24

18.3

24

19.7

36.8

19.8

17.3–18.0

33.3

54

19.9

37.3

23

28.1

50.6

6.3

20.7

anteiso-C15:0

7.4

4.5

2.5

4.5

tr

2.1–2.8

7.2

1.2

tr

4.5

iso-C16:0

1.2

1.1

1.2

1.1

tr

3.7

1.9

1.1–1.2

tr

1

2.6

iso-C15:0 2-OH/C16:1ω7c

5.3

2.7

1.7

13.7

1.7

22.8

0.8

34.8

30.7–30.8

9.9

0.5

10

23.2

1.5

9.2

C16:1ω5c

21.3

17.5

23.4

21.6

6.7–7.8

11

2.8

37.7

2.3

21

9.9

4.9

44.8

3.3

iso-C15:0 3-OH

5.1

3.4

3

12.5

3

2.8

3.4–5.0

1

3.2

4

4

2

3.7

2.2

4.3

2.2

anteiso-C17:1 B/i I

1.2

iso-C16:0 3-OH

1

4.1

12.7

4.1

tr

2.8

tr

0.6–0.7

1.3

0.5

tr

1.9

C16:0 3-OH

3.2

1.9

1.6

1.2

1.6

1.2

0.9–1.4

7.6

4.1

2.8

8.6

18

4.9

1.1

iso-C17:0 3-OH

9.8

12.4

7.7

9.3

7.7

1.9

12.2

4.1

9.4–10.0

5

12.2

7.1

0.3

2

4.5

2.4

10.8

3.2

Strains: 1 N5DB8-4T (Tunicatimonas pelagia gen. nov., sp. nov.; present study), 2Porifericola rhodea N5EA6-3A2BT (Yoon et al. 2011), 3Roseivirga ehrenbergii KMM 6017T (Nedashkovskaya et al. 2005a, b, c), 4Fabibacter halotolerans UST030701-084T (Lau et al. 2006), 5Fulvivirga kasyanovii KMM 6220T (Nedashkovskaya et al. 2007a, b), 6Reichenbachiella agariperforans (Nedashkovskaya et al. 2003), 7Marivirga tractuosa NBRC 15989T (Nedashkovskaya et al. 2010), 8Marinoscillum pacificum MRN461T (Seo et al. 2009), 9Echinicola pacifica KMM 6172T (Nedashkovskaya et al. 2006), 10Aureibacter tunicatorum A5Q-118T (Yoon et al. 2010), 11Persicobacter diffluens NBRC 15940T (Muramatsu et al. 2010), 12Flexithrix dorothea ATCC 23163T (Lewin 1970), 13Flammeovirga aprica NBRC 15941T (Takahashi et al. 2006), 14Sediminitomix flava Mok-1-85T (Khan et al. 2007), 15Limibacter armeniacum YM11-185T (Yoon et al. 2008), 16Perexilibacter aurantiacus Shu-F-UV2-2T (Yoon et al. 2007), 17Rapidithrix thailandica TISTR 1750T (Srisukchayakul et al. 2007), 18Belliella baltica BA134T (Brettar et al. 2004)

Values are percentages of total fatty acids

– Not detected, tr traces (<1%)

Polyphasic taxonomic conclusion

From the distinct phylogenetic position and combinations of genotypic (16S rRNA gene sequences similarity) and phenotypic (cell morphology, length, colony color, gliding motility and pigments production) characteristics, strain N5DB8-4T cannot be assigned to any previously recognized bacterial genus and thus can be described as a novel species within a new genus, Tunicatimonas pelagia gen. nov., sp. nov.

Description of Tunicatimonas gen. nov.

Tunicatimonas [Tu.ni.ca.ti.mo’nas. N.L. pl. n. Tunicata, scientific name of a subphylum (tunicates); L. fem. n. monas, a unit, monad; N.L. fem. n. Tunicatimonas, a unit (bacterium) isolated from tunicates].

A member of the family Flammeovirgaceae, phylum Bacteroidetes, according to 16S rRNA analyses. Cells are straight-rod-shaped, Gram-stain negative and strictly aerobic. Endospores are not formed. Catalase-positive, but oxidase-negative. Flexirubin-type pigments are absent. The major respiratory quinone is menaquinone 7 (MK-7). The predominant cellular fatty acids are iso-C15:0, C16:1ω5c and iso-C15:1 G. The DNA G+C content of the type strain of the type species is 52.6 mol%.

The type species is Tunicatimonas pelagia.

Description of Tunicatimonas pelagia sp. nov.

Tunicatimonas pelagia (pe.la’gi.a. L. fem. adj. pelagia, of the sea).

The main characteristics are the same as those given for the genus. In addition, cells are straight-rods 0.3–0.5 μm in width and 15–30 μm in length. Cells lack flagella and are non-motile. Gliding motility is not observed. Colonies grown on marine agar 2216 are 1–2 mm in diameter, circular, shiny with entire edges and reddish-pink-pigmented. Colonies are pink but become reddish-pink after two weeks of incubation. Temperature range for growth is 20–37°C, the optimal temperature is between 25 and 30°C, but no growth occurs at 4 or 45°C. The pH range for growth is 6–10 (optimum, pH 7), while no growth was observed below 6 or above 10. NaCl is required for growth and can be tolerated at a concentration of up to 8% (w/v). No growth was occurred above 9% (w/v) NaCl. Nitrate and nitrite reduction are negative. Agar, casein, DNA, gelatin, starch, tyrosine, Tweens 20, Tweens80 and urea are not hydrolysed. The o-nitrophenyl-β-d-galactosidase (ONPG) test is positive but the reactions for the Voges-Proskauer test, citrate utilization, arginine dihydrolase, lysine decarboxylase and ornithine decarboxylase activities and hydrogen sulfide and indole production are negative. Acid production tests were carried out using API50CH strips. Acid is produced from d-arabinose, l-arabinose, d-xylose, l-xylose, methyl-β-d-xylopyranoside, galactose, glucose, fructose, mannose, rhamnose, methyl-α-d-mannopyranoside, methyl-α-d-glucopyranoside and N-acetyl-glucosamine but not from amygdalin, arbutin, esculin ferric citrate, salicin, cellobiose, maltose, lactose, melibiose, sucrose, trehalose, melezitose, d-turanose, d-lyxose, d-tagatose, d-fucose, l-fucose, inulin, ribose, raffinose, gentiobiose, glycerol, erythritol, adonitol, sorbose, dulcitol, inositol, mannitol, sorbitol, starch, glycogen, xylitol, d-arabitol, l-arabitol, gluconate, 2-keto-gluconate or 5-keto-gluconate. Alkaline phosphatase, esterase (C4), esterase lipase (C8), lipase (C4), leucine arylamidase, valine arylamidase, cystine arylamidase, α-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase and N-acetyl-β-glucosaminidase activities are present but α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, trypsin, β-glucuronidase, α-mannosidase, and α-fucosidase activities are absent. The major (>1.0%) fatty acids are iso-C13:0 (9.8), iso-C15:1 G (11.3), iso-C15:0 (24.5), iso-C15:0 2-OH/C16:1ω7c (5.3), C16:1ω5c (21.3), C16:0 (1.4), iso-C15:0 3-OH (5.1), C16:0 3-OH (3.2) and iso-C17:0 3-OH (9.8). The G+C of the genomic DNA is 52.6 mol%.

The type strain is N5DB8-4T (= KCTC 23473T = NBRC 107804T), which was isolated from an orange-striped sea anemone Diadumene lineate in Chiba Prefecture, Japan.

Notes

Acknowledgment

The present research has been conducted by the Settlement Research Grant of Keimyung University in 2011.

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Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Jaewoo Yoon
    • 1
  • Naoya Oku
    • 2
    • 4
  • Sanghwa Park
    • 3
  • Atsuko Katsuta
    • 2
    • 5
  • Hiroaki Kasai
    • 2
    • 5
  1. 1.College of PharmacyKeimyung UniversityDaeguRepublic of Korea
  2. 2.Marine Biotechnology Institute Co. LtdIwateJapan
  3. 3.Institute of Molecular and Cellular BiosciencesThe University of TokyoTokyoJapan
  4. 4.Biotechnology Research CenterToyama Prefectural UniversityToyamaJapan
  5. 5.Marine Biosciences Kamaishi Research LaboratoryKitasato UniversityIwateJapan

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