Antonie van Leeuwenhoek (2015) 107:1607–1613 DOI 10.1007/s10482-015-0456-9

ORIGINAL PAPER

Wenyingzhuangia gracilariae sp. nov., a novel marine bacterium of the phylum Bacteroidetes isolated from the red alga Gracilaria vermiculophylla Jaewoo Yoon • Naoya Oku • Hiroaki Kasai

Received: 13 March 2015 / Accepted: 11 April 2015 / Published online: 21 April 2015 Ó Springer International Publishing Switzerland 2015

Abstract A Gram-negative, strictly aerobic, beigepigmented, non-motile, rod-shaped bacterial strain designated N5DB13-4T was isolated from the red alga Gracilaria vermiculophylla (Rhodophyta) collected at Sodegaura Beach, Chiba, Japan. Phylogenetic analyses based on the 16S rRNA gene sequence revealed that the novel isolate is affiliated with the family Flavobacteriaceae within the phylum Bacteroidetes and that it showed highest sequence similarity (97.3 %) to Wenyingzhuangia heitensis H-MN17T. The hybridization values for DNA–DNA relatedness between the strains N5DB13-4T and W. heitensis H-MN17T were 34.1 ± 3.5 %, which is below the threshold accepted for the phylogenetic definition of a

novel prokaryotic species. The DNA G?C content of strain N5DB13-4T was determined to be 31.8 mol%; MK-6 was identified as the major menaquinone; and the presence of iso-C15:0, iso-C15:0 3-OH and isoC17:0 3-OH as the major ([10 %) cellular fatty acids. A complex polar lipid profile was present consisting of phosphatidylethanolamine, two unidentified glycolipids and four unidentified lipids. From the distinct phylogenetic position and combination of genotypic and phenotypic characteristics, the strain is considered to represent a novel species of the genus Wenyingzhuangia for which the name Wenyingzhuangia gracilariae sp. nov. is proposed. The type strain of W. gracilariae sp. nov. is N5DB13-4T (=KCTC 42246 T =NBRC 110602T).

Electronic supplementary material The online version of this article (doi:10.1007/s10482-015-0456-9) contains supplementary material, which is available to authorized users.

Keywords Bacteroidetes  Flavobacteriaceae  Wenyingzhuangia gracilariae sp. nov.  Rhodophyta  16S rRNA gene  Polyphasic taxonomy

J. Yoon (&) College of Pharmacy, Keimyung University, 1095 Dalgubeoldaero, Dalseo-Gu, Daegu 704-701, Republic of Korea e-mail: [email protected] N. Oku Biotechnology Research Center, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan H. Kasai Marine Biosciences Kamaishi Research Laboratory, Kitasato University, 160-4 Utou, Okirai, Sanriku-cho, Ofunato, Iwate 022-0101, Japan

Introduction A wide array of molecular phylogenetic studies based on 16S rRNA gene sequences have shown that species of the phylum Bacteroidetes are widespread in nature including terrestrial, marine environments and sediments (DeLong et al. 1993; Bowman et al. 1997; Glo¨ckner et al. 1999; O’Sullivan et al. 2002). The family Flavobacteriaceae (Reichenbach 1989; Bernardet et al. 2002) is one of the major phylogenetic

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groups within the phylum Bacteroidetes and is comprised of more than 120 validly named genera (www. bacterio.cict.fr/f/flavobacteriaceae.html). Several species of the family often colonize the surface of the coastal red algae (Nylund et al. 2010; Fernandez et al. 2012; Miranda et al. 2013) and are known to be involved in decomposition of various bioorganic macromolecules such as agar, cellulose, chitin and pectin (Bianchi and Bianchi 1995; Cottrell and Kirchman 2000; Kirchman 2002; Pinhassi and Berman 2003; Brettar et al. 2004). However, there have been relatively few studies of their detailed ecophysiology, biochemistry and taxonomy. The genus Wenyingzhuangia, a member of the family Flavobacteriaceae of the phylum Bacteroidetes, was first proposed by Liu et al. (2014). At the time of writing, the genus Wenyingzhuangia includes the two marine species, Wenyingzhuangia marina D1T (Liu et al. 2014) and Wenyingzhuangia heitensis H-MN17T (Yoon and Kasai 2015), which have been isolated from a recirculating mariculture system and seawater, respectively. In this study, we have characterised a novel marine Bacteroidetes strain, N5DB13-4T, isolated from the red alga Gracilaria vermiculophylla (Rhodophyta), by using polyphasic taxonomic methods, including 16S rRNA gene sequence analysis and physiological, biochemical and chemotaxonomic analyses. Based on the polyphasic taxonomic data, we suggest that the isolate represents a novel species of the genus Wenyingzhuangia within the phylum Bacteroidetes.

Materials and methods Isolation of the bacterial strain and culture conditions Strain N5DB13-4T was isolated from the red alga G. vermiculophylla (Rhodophyta) collected at Sodegaura Beach, Chiba, Japan in August 2005 (GPS location: N: 35°270 17.000 , E: 139°570 63.800 ). A piece of the algae (approximately 1 g) was gently rinsed in sterile artificial seawater and then homogenised with a sterilised glass rod. After leaving the homogenate for 1 h until the most of suspended material settled out, a 50 lL portion of the supernatant was spread onto a plate (/ 9 cm) containing 1/10 MA-Con.A [marine agar 2216 (Difco) 5.51 g/L, 75 % natural seawater;

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supplemented with powdered concanavalin A (a lectin from jack bean Canavalia ensiformis) 10 mg/L after the autoclaved mixture was cooled to 80 °C]. The plate was left at ambient temperature (20–28 °C) for 2 weeks and the beige-coloured colonies that grew were purified on fresh marine agar 2216. The strain was routinely subcultured on marine agar 2216 at 28 °C and maintained in marine broth 2216 (Difco) supplemented with 20 % (v/v) glycerol at 70 °C. Morphological, physiological and biochemical analysis Cell morphology was observed by using transmission electron microscopy (TEM) and motility was measured by phase contrast microscopy (Primo Star; ZEISS). Gliding motility was determined as described by Perry (1973). For TEM, cells were mounted on Formvar-coated copper grids and negatively stained with 0.75 % (w/v) uranyl formate. Grids were observed using a Tenai G2 spirit (FEI) microscope operated at 120 kV at a magnification of 15,000. The temperature range (4, 10, 15, 20, 30, 37, 40 and 45 °C) and pH range (5.5–9.5) for colony growth were determined by incubating the isolate for 2 weeks on marine agar 2216. The following buffers (Sigma-Aldrich Buffer Reference Center, http://www.sigmaaldrich. com/life-science/core-bioreagents/biological-buffers/ learning-center/buffer-reference-center.html) were used for pH tests: MES (pH 5.5), ACES (pH 6.5 and 7.0), TAPSO (pH 7.6), TAPS (pH 8.5) and CHES (pH 9.0 and 9.5). The NaCl concentration range for growth was determined on TY agar medium [0.2 % tryptone, 0.1 % yeast extract and 1.5 % agar (w/v) with 0–10 % (w/v) NaCl] and the cells were grown at 28 °C. Gramstaining was performed using the BD Gram Staining Kit (Becton, Dickinson and Company, USA). Spore formation was tested by staining with malachite green. Anaerobic growth was tested for up to 2 weeks on marine agar 2216 in a jar containing AnaeroPackAnaero (Mitsubishi Gas Chemical Co, Inc), which works as an O2 absorber and CO2 generator. Catalase activity was detected by the observation of the formation of bubbles in 3 % (v/v) H2O2 solution. Oxidase activity test was performed using commercial dropper oxidase (Becton, Dickinson and Co). Degradation of DNA was tested using DNase agar (Scharlau Chemie) (Collins and Lyne 1984), with DNase activity detected

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by flooding plates with 1 M HCl. The ability to hydrolyse casein, Tween 20, Tween 80 and tyrosine was determined according to Hansen and Sørheim (1991). API 20E, API 50CH and API ZYM strips (bioMe´rieux) 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, API 50CH and API ZYM test strips were read after 72 h incubation at 28 °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 DNA G?C content, 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 1500 bp long 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 N5DB13-4T was compared with sequences obtained from GenBank (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov). The EzTaxon-e database (Kim et al. 2012) was used to identify the nearest taxa. 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 1348 bases of the 16S rRNA gene were compared. Evolutionary distances (distance options according to Kimura’s two-parameter model; Kimura 1983) were calculated and clustering was performed with the neighbourjoining method (Saitou and Nei 1987), maximumparsimony (Fitch 1971) and maximum-likelihood (Felsenstein 1985) methods using MEGA5 software (Tamura et al. 2011). Bootstrap analysis was used to evaluate the tree topology of the neighbour-joining data by performing 1000 resamplings (Felsenstein 1985). The topology of the phylogenetic tree was evaluated by the bootstrap resampling method of Felsenstein (1985) with 1000 replicates.

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DNA–DNA hybridization test DNA–DNA hybridization was performed by the membrane filter method (Akagawa-Matsushita et al. 1992). Each mixture of labeled and unlabeled DNAs was incubated at 37 °C for 12 h. Reciprocal hybridization tests were performed in triplicate. Chemotaxonomic analysis Gas chromatography analysis of the cellular fatty acid methyl esters was performed using a culture grown on marine agar 2216 at 28 °C for 3 days and fatty acid methyl esters were extracted and prepared according to standard protocols provided by the MIDI/Hewlett Packard Microbial Identification system Sherlock Version 3.10/TSBA 50 (Sasser 1990). Polar lipids were extracted according to the procedures described by Minnikin et al. (1984). They were identified by two-dimensional TLC followed by spraying with appropriate detection reagents (Minnikin et al. 1984; Komagata and Suzuki 1987). Phospholipids were detected with the Zinzadze reagent of Dittmer and Lester (1964). Whole lipid profiles were detected by spraying with molybdatophosphoric acid (5 g molybdatophosphoric acid hydrate in 100 ml ethanol) followed by heating at 150 °C (Worliczek et al. 2007). Determination of the respiratory quinone system was carried out as described previously (Collins and Jones 1981).

Results and discussion Morphological, physiological and biochemical characteristics Cells of strain N5DB13-4T grown on marine agar 2216 were observed to be straight rods with 0.8–0.9 lm in width and 2.0–2.3 lm in length, devoid of flagella or cell appendages (Fig. 1) and to produce a beige pigment. Gliding motility was not observed by light microscopy. Flexirubin-type pigments were not produced. The strain showed distinct phenotypic, physiological and biochemical features that discriminate it from the closest related member in the genus Wenyingzhuangia as shown in Table 1.

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Fig. 1 Transmission electron micrograph of a negatively stained cell of strain N5DB13-4T. Bar 1 lm

Phylogenetic analysis and DNA–DNA hybridization test The almost complete 16S rRNA gene sequence was determined for strain N5DB13-4T (GenBank/

EMBL/DDBJ accession number AB983340). An evolutionary tree based on the neighbour-joining method generated a comparison of the 16S rRNA gene sequences and showed that strain N5DB13-4T is phylogenetically affiliated with the members of the genus Wenyingzhuangia of the phylum Bacteroidetes (Fig. 2). Comparative analysis of 16S rRNA gene sequences revealed that the sequence of strain N5DB13-4T had a similarity of 97.3 % to that of W. heitensis H-MN17T and 96.5 % to W. marina D1T. All other cultivated species of the phylum Bacteroidetes with validly published names were found to be more distantly related, showing a 16S rRNA gene sequence similarity of less than 91 %. DNA–DNA hybridization values between strain N5DB13-4T and W. marina D1T were 34.1 ± 3.5 %. These results strongly suggest that strain N5DB13-4T should be classified as a separate species, if it can be distinguished by phenotypic traits (Wayne et al. 1987).

Table 1 Differential characteristics of strain N5DB13-4T and other closely related taxa Characteristic

1

2

3

Cell morphology

Rods

Rods

Rods or coccoids

Pigmentation

Beige

Beige

Yellow

Length

2.0–2.3

2.3–2.5

0.5–1.0

Width

0.8–0.9

0.4–0.5

0.3–0.5

20–30

25–30

15–30

Cell size (lm)

Temperature range for growth (°C) Highest NaCl tolerance (%, w/v)

6

5

8

Catalase

?

-

?

Agar

-

?

-

Starch

-

-

?

Cellobiose Galactose

?

-

? ?

Glucose

-

-

?

Maltose

-

-

?

Methyl-b-D-xylopyranoside

?

?

-

Hydrolysis of

Acid production from

Polar lipids

PE, 2UGL, 4UL

PE, 3UAL, 3UGL, 3UL

PE, 4UL

DNA G?C content (mol%)

31.8

31.8

35.9

Strains: 1 N5DB13-4T (Wenyingzhuangia gracilariae; present study), 2 Wenyingzhuangia heitensis H-MN17T (Yoon and Kasai 2015), 3 Wenyingzhuangia marina D1T (Liu et al. 2014) PE phosphatidylenthanolamine, UAL unidentified aminolipid, UGL unidentified glycolipid, UL unidentified lipid, ? positive, negative

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1611 Polaribacter irgensii 23-PT (M61002)

44/80/59

Polaribacter butkevichii KMM 3938T (AY189722)

97/80/95 91/94/74 83/59/60 100/99/99

Polaribacter franzmannii 301T (U14586) Polaribacter filamentus 215T (U73726)

Polaribacter dokdonensis DSW-5T (DQ004686) Polaribacter gangjinensis K17-16T (FJ425213)

70/93/65

52/67/45 27/25/52 38/67/25 100/99/100

Lutibacter litoralis CL-TF09T (AY962293) Lutibacter flavus IMCC1507T (GU166749) Lutibacter agarilyticus KYW566T (JN864028) Lutibacter maritimus S7-2T (FJ598048) Lutibacter aestuarii MA-My1T (HM234096)

60/100/57 100/83/100

Wenyingzhuangia marina D1T (JQ948039) Wenyingzhuangia heitensis H-MN17T (AB924513)

Wenyingzhuangia gracilariae N5DB13-4T (AB983340) Flammeovirga aprica NBRC 15941T (AB247553)

Fig. 2 Neighbour-joining tree of 16S rRNA gene sequence similarity, showing the phylogenetic position of strain N5DB134T and representatives of closely related and other more distantly related taxa in the phylum Bacteroidetes. The tree was rooted using Flammeovirga aprica NBRC 15941T (AB247553) as an outgroup. The numbers at the nodes indicate

the percentages of the occurrence of the strain in 1000 bootstrapped trees. The sequence determined in this study is shown in bold. Bootstrap values from neighbour-joining, maximum-parsimony and maximum-likelihood analyses are shown (NJ/MP/ML). Bar 2 % sequence divergence

Chemotaxonomic characteristics

Polyphasic taxonomic conclusion

As shown in Table 2, the predominant cellular fatty acids ([10 %) of strain N5DB13-4T were identified as iso-C15:0 (17.3 %), iso-C15:0 3-OH (18.3 %) and isoC17:0 3-OH (18.9 %) as identified by the MIDI system. On the basis of the fatty acid composition, strain N5DB13-4T could be differentiated from the phylogenetically closely related taxa W. marina D1T and W. heitensis H-MN17T as shown in Table 2. Moreover, strain N5DB13-4T could be distinguished from the species W. marina D1T and W. heitensis H-MN17T by the presence of C20:4 x6,9,12,15c (Table 2). The polar lipids of strain N5DB13-4T were determined to be composed of phosphatidylethanolamine, two unidentified glycolipids and four unidentified lipids (Supplementary Fig. 1). The two unidentified glycolipids and four unidentified lipids distinguished strain N5DB13-4T from the other species in the genus Wenyingzhuangia. From these results, it is suggested that strain N5DB13-4T represents an independent species of the genus Wenyingzhuangia of the family Flavobacteriaceae within the phylum Bacteroidetes (Table 1).

From the distinct phylogenetic position and combinations of genotypic and phenotypic characteristics, strain N5DB13-4T cannot be assigned to any previously recognised species in the genus Wenyingzhuangia and thus can be described as representing a novel species, Wenyingzhuangia gracilariae sp. nov.

Description of Wenyingzhuangia gracilariae sp. nov Wenyingzhuangia gracilariae (gra.ci.la’ri.ae. N.L. fem. gen. n. gracilariae of the red alga Gracilaria). Cells are straight-rods, 0.8–0.9 lm in width and 2.0–2.3 lm in length. Cells lack flagella and are nonmotile. Gliding motility is not observed. Colonies grown on marine agar 2216 are circular and beige pigmented after 5 days of incubation at 28 °C. Temperature range for growth is 20–30 °C; the optimal temperature is around 28 °C but no growth occurs at 4 or 45 °C. The pH range for growth is 6–9 (optimum, pH 7), while no growth is observed below 6 or above 9. NaCl is required for growth and can be tolerated at a

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Table 2 Comparison of cellular fatty acids for strain N5DB13-4T and other closely related taxa Fatty acid

1

2

3

iso-C13:0

5.8

tr

6.1

iso-C14:0

–

tr

–

C15:0 3-OH

–

–

1.6

iso-C15:0

17.3

4.1

23.2

iso-C15:0 3-OH

18.3

–

13.5

iso-C15:1 G

5.7

–

10.8

anteiso-C15:0

tr

37.9

1.4

C15:1 x6c

tr

–

1.5

C16:0

2.7

tr

2

C16:0 3-OH

3.1

–

1.9

iso-C16:0

tr

tr

–

iso-C17:0

tr

8.8

–

iso-C17:0 3-OH

18.9

–

15.2

anteiso-C17:0

tr

19.1

–

C18:0 iso-C18:0

tr tr

3 1.2

– –

C19:0

–

tr

–

iso-C19:0

–

8

–

anteiso-C19:0

–

11.8

–

C20:0

–

2.5

–

C20:4 x6,9,12,15c

5.8

–

–

a

8.9

–

14.3

Summed feature 3

The data were typically obtained by GLC using the MIDI system Strains: 1 N5DB13-4T (Wenyingzhuangia gracilariae sp. nov.; present study), 2 Wenyingzhuangia heitensis H-MN17T (Yoon and Kasai 2015), 3 Wenyingzhuangia marina D1T (Liu et al. 2014) tr trace (less than 1.0 %), – not detected a

Summed feature 3 consists of C16:1 x7c and/or C16:1 x6c

concentration of up to 5 % (w/v) but no growth occurs above 6 % (w/v) NaCl. Nitrate is not reduced. Agar, casein, DNA, gelatin, starch, tyrosine, Tween 20, Tween 80 and urea are not hydrolysed. The reaction for o-nitrophenyl-b-D-galactopyranoside (ONPG) is positive but arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, citrate utilisation, hydrogen sulfide production, indole production and Voges–Proskauer test are negative (API 20E). Acid production tests using API 50CH strips give the following reactions: acid is produced from adonitol, methyl-b-D-xylopyranoside, galactose, esculin ferric citrate, salicin, lactose, melibiose, D-turanose and Dlyxose but not from glycerol, N-acetylglucosamine,

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arbutin, 5-keto-gluconate, D-arabinose, glucose, fructose, mannose, L-arabitol, amygdalin, maltose, sucrose, trehalose, starch, glycogen, gentiobiose, Lfucose, ribose, sorbose, rhamnose, sorbitol, methyla-D-mannopyranoside, L-arabinose, D-xylose, L-xylose, methyl-a-D-glucopyranoside, cellobiose, melezitose, D-tagatose, D-fucose, inulin, raffinose, erythritol, dulcitol, inositol, mannitol, xylitol, Darabitol, gluconate and 2-keto-gluconate. In the API ZYM strip, alkaline phosphatase, leucine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase and a-galactosidase are present but b-glucosidase, afucosidase, valine arylamidase, trypsin, b-galactosidase, a-glucosidase, N-acetyl-b-glucosaminidase, esterase (C4), esterase lipase (C8), lipase (C4), cystine arylamidase, a-chymotrypsin, b-glucuronidase and amannosidase are absent. The major ([10 %) fatty acids are iso-C15:0, iso-C15:0 3-OH and iso-C17:0 3-OH. The major polar lipids are phosphatidylethanolamine, two unidentified glycolipids and four unidentified lipids. The G?C of the genomic DNA of the type strain is 31.8 mol%. The type strain is N5DB13-4T (=KCTC 42246 T =NBRC 110602T), which was isolated from Gracilaria vermiculophylla (Rhodophyta) collected at Sodegaura Beach, Chiba, Japan. The GenBank/ EMBL/DDBJ accession number of the 16S rRNA gene sequence of strain N5DB13-4T is AB983340. Acknowledgments This work was supported by the New Energy and Industrial Technology Development Organization (NEDO), and in part by a research grant (2009–2011) of the Institute for Fermentation, Osaka, Japan. Conflict of interest The authors declare that they have no conflict of interest.

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